Latest revision as of 22:53, 13 October 2018

Error Prone PCR · Benchling

Error Prone PCR

Introduction

Error prone PCR is a method by which random mutants may be inserted into any piece of DNA.

Materials

- DNA primers for PCR amplification
- DNA template
- Four separate solutions of 20 mM dATP, TTP, dCTP, and dGTP
- Taq DNA polymerase, stored at -20 C
- 10 X PCR buffer: 100 mM Tris-HCl, pH 8.3, 15 mM MgCl2, and 500 mM KCl
- 1 MgCl2
- 50 mM MnCl2
- Agarose
- 10 X TBE buffer: 1 M Tris base, 1 M boric acid, and 20 mM EDTA, pH 8.0
- 10 mg/mL ethidium bromide
- Loading buffer: 10 mM Tris-HCl, pH 7.6, 0.15 % orange G, 0.03% xylene cyanol FF, 60% glycerol, 60 mM EDTA
- DNA mass ladder for quantification
- Gel purification kit

Procedure

Prepare a stock solution of the DNA template (~50-100 ng/μL) in water.

Label 16 thin-walled PCR tubes as "reactions 1 through 16".

Combine the reagents listed in Table 1 in a conical vial and label it "PCR reaction mixture"

Dispense the PCR reaction mixture from step 3 into tubes 1 through 16 by 96 μL to tube 1 and add 88 μL to tubes 2-16.

Add 2 μL of the DNA template to tube 1.

Place tube 1 in the thermocycler and start the PCR program (Table 2).

	A A	B B	C C	D D
1 1	Reagents	Stock Concentration	Volume	Final Concentration
2 2	Forward Primer	100 μM	15 μL	1 μM
3 3	Reverse Primer	100 μM	15 μL	1 μM
4 4	dCTP and dTTP	20 mM	75 μL/ea	1 mM
5 5	dATP and dGTP	20 mM	15 μL/ea	0.2 mM
6 6	PCR buffer w/ Mg2+	10X	150 μL	1x
7 7	MgCl2	1M	8 μL	~5.5 mM
8 8	DMDC H2O		1098 μL
9 9	Final Volume		1491 μL

PCR Reagent Mixture

	A A	B B	C C	D D
1 1	Step	Temperature	Duration
2 2	Template Denaturation	95 - 98°C	1 min	One time only
3 3	Oligo Denaturation	94°C	1 min	Repeat next 3 steps x30
4 4	Annealing	58°C	1 min	This may change depending on primers
5 5	Extension	72°C	2 min
6 6	Final Elongation	72°C	5 min
7 7	Product Storage	4°C	forever	Repeat steps 3-5 for 30 cycles

Table1

Once the PCR program has reached the annealing temperature, add 1 μL of freshly prepared MnCl2 solution and 1 μL of Taq DNA polymerase to the PCR reaction tube.

Perform 4 cycles of PCR amplification using the hot start procedure (see Note 3).

Remove the PCR tube from the thermocycler

Place the tube on ice.

Transfer 10 μL of the PCR reaction from tube 1 to tube 2.

Place tube 2 in the thermocycler and start the PCR program.

Once the PCR program has reached the annealing temperature, add 1 μL of freshly prepared MnCl2 and 1 μL of Taq DNA polymerase to the PCR reaction tube.

Perform 4 cycles of PCR amplification using the host start procedure.

Remove the PCR tube from the thermocycler

Place the tube on ice.

Repeat steps 11 through 16 using tubes 3 through 16 to create a mutagenic library by serial dilution amplification (image down below)

Verify the quality of each PCR reaction by agarose gel electrophoresis.

Combine 50 μL of each PCR reaction into a single tube and store on ice; this is your DNA library

Purify the DNA library by agarose gel electrophoresis

Recover the DNA library using a gel purification kit.

Quantify the DNA library by agarose gel electrophoresis

NOTES

Primer should be complemetary and have similar melting techniques: http://bioweb.uwlax.edu/GenWeb/Molecular/seq_anal/primer_design/primer_design.htm

Optimal PCR conditions should be determined prior to the use of this mutagenic PCR protocol. The doubling efficiency for the normal PCR reaction should be ∼1.7–1.9 per PCR cycle and can be analyzed by running the PCR product after each cycle on an agarose gel

Taq DNA polymerase should be stored at −20°C and kept on ice or in a frozen metal block when taken out of the freezer. The hot start procedure refers to adding the Taq DNA polymerase to the PCR reaction mixture after the mixture has reached the annealing temperature. Either standard or “hot start” Taq DNA polymerase will work in the described method.

It is important to prepare a fresh MnCl₂ solution for the error-prone PCR experiment. This solution should be stored on ice and combined with the PCR reaction mixture at the start of each serial dilution.

It is important to prepare a fresh MnCl₂ solution for the error-prone PCR experiment. This solution should be stored on ice and combined with the PCR reaction mixture at the start of each serial dilution.

The method described here was optimized for a 400-nucleotide gene. We highly recommend that experimenters read ref. 7 for information on the predicted average number of doublings and mutations expected as a function of template length

Agarose gel electrophoresis should be performed by standard methods. A DNA ladder with an appropriate base pair range should be used to identify and estimate the quantity of the PCR product.

It is recommended to save a portion of the PCR product from each tube for further analysis or amplification.

The concentration of the DNA product can be determined by comparing the intensity of the product bands to the intensity of the bands from the DNA quantification ladder from commercial sources.

Transformation of Libraries

GIbson Assembely can be performed if the primers of the linearized vector are complimentery to the error-prone PCR product.

References

Random Mutagenesis by Error-Prone PCR McCullum E Williams B Zhang J Chaput J Methods in molecular biology (Clifton, N.J.) 2010 vol: 634 pp: 103-109

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 </head>
 <body><div><svg id="__SVG_SPRITE_NODE__" style="position: absolute; width: 0; height: 0" xmlns="http://www.w3.org/2000/svg" xmlns:xlink="http://www.w3.org/1999/xlink"></svg><div><div data-reactroot=""><div class="printable-container hide-print-links"><div><div class="protocolEditor"><div><!-- react-empty: 6 --><!-- react-empty: 7 --></div><div class="protocolEditor-pageContainer"><div class="protocolEditor-page"><div class="protocolEditor-contentWrapper"><h1 class="mediocre-titleEditor is-locked"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="false" data-cid="pv887-title" data-index="0">Error Prone PCR</div></h1><div><h2>Introduction</h2><div class="intro-editor" data-editor="intro"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="true" data-cid="pv887-intro" data-index="0"><b><i><span style="color:#6A6A6A"><span style="background-color:#FFFFFF">Error prone PCR</span></span></i></b><span style="color:#545454"><span style="background-color:#FFFFFF"> is a method by which random mutants may be inserted into any piece of DNA.</span></span>
-</div></div><h2>Materials</h2><div class="materials-editor" data-editor="materials"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><ul class="materials"><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m888" data-index="0"></div><ul class="components"><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c889" data-index="0,0">DNA primers for PCR amplification</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c890" data-index="0,1">DNA template</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c891" data-index="0,2">Four separate solutions of 20 mM dATP, TTP, dCTP, and dGTP</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c892" data-index="0,3"><i>Taq </i>DNA polymerase, stored at -20 C</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c893" data-index="0,4">10 X PCR buffer: 100 mM Tris-HCl, pH 8.3, 15 mM MgCl2, and 500 mM KCl</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c894" data-index="0,5">1 MgCl2</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c895" data-index="0,6">50 mM MnCl2</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c896" data-index="0,7">Agarose</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c897" data-index="0,8">10 X TBE buffer: 1 M Tris base, 1 M boric acid, and 20 mM EDTA, pH 8.0</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c898" data-index="0,9">10 mg/mL ethidium bromide </div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c899" data-index="0,10">Loading buffer: 10 mM Tris-HCl, pH 7.6, 0.15 % orange G, 0.03% xylene cyanol FF, 60% glycerol, 60 mM EDTA</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c900" data-index="0,11">DNA mass ladder for quantification</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c901" data-index="0,12">Gel purification kit </div></li></ul></li></ul></div><h2>Procedure</h2><div class="stepsEditor mediocre"><div class="editor" data-editor="steps"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><div class="mediocre-item is-header" data-scroll-id="s902"><ul class="headers"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s902" data-index="0"></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s902" data-index="0" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ul></div><div class="mediocre-item is-step" data-scroll-id="s903"><ol class="steps"><li value="1"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s903" data-index="1">Prepare a stock solution of the DNA template (~50-100 ng/<span style="color:#333333"><span style="background-color:#FFFFFF">μL) in water. </span></span></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s903" data-index="1" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s904"><ol class="steps"><li value="2"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s904" data-index="2">Label 16 thin-walled PCR tubes as "reactions 1 through 16".</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s904" data-index="2" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s905"><ol class="steps"><li value="3"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s905" data-index="3">Combine the reagents listed in Table 1 in a conical vial and label it "PCR reaction mixture"</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s905" data-index="3" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s906"><ol class="steps"><li value="4"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s906" data-index="4">Dispense the PCR reaction mixture from <b>step 3 </b>into tubes 1 through 16 by 96 <span style="color:#344043"><span style="background-color:#FFFFFF">μL to tube 1 and add 88 </span></span><span style="color:#344043"><span style="background-color:#FFFFFF">μL to tubes 2-16. </span></span></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s906" data-index="4" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s907"><ol class="steps"><li value="5"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s907" data-index="5">Add 2 <span style="color:#344043"><span style="background-color:#FFFFFF">μL of the DNA template to tube 1.</span></span></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s907" data-index="5" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s908"><ol class="steps"><li value="6"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s908" data-index="6">Place tube 1 in the thermocycler and start the PCR program (Table 2).</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s908" data-index="6" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-table" data-scroll-id="s909"><div class="mediocre-tableEditable printable-container-scaleForPrint" data-formattable="true" data-index="7" data-subindex="cell"><div><!-- react-empty: 113 --><!-- react-empty: 114 --><!-- react-empty: 115 --></div><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><div class="hiddenFocusEditable editable hiddenFocusEditable--fullTable" contenteditable="true" data-cid="s909-table" data-formattable="true" data-index="7" data-subindex="table"></div><div class="u-position-relative mediocre-tableEditable-scrollAreaWrapper" style="margin-left: 0px; margin-right: 0px;"><div class="mediocre-tableEditable-scrollArea" style="padding-top: 41px;"><div class="mediocre-tableEditable-tableWrapper" style="padding-left: 0px;"><div class="mediocre-tableEditable-border multi-editable" style="left: -6px; top: -37px; bottom: 4px; width: 457px; min-width: 12px;"></div><div class="u-position-relative u-flex-display"><table class="mediocre-tableEditable-table mediocre-tableEditable-table--main" style="width: 445px;"><colgroup><col style="width: 45px;"></col><col data-id="0" style="width: 100px;"></col><col data-id="1" style="width: 100px;"></col><col data-id="2" style="width: 100px;"></col><col data-id="3" style="width: 100px;"></col></colgroup><tbody><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-maximizeButton"><span class="glyphicon glyphicon-arrow-up mediocre-tableEditable-maximizeIcon" icon="arrow-up"></span></td><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--col"><div class="mediocre-tableEditable-axisCell-labelWrapper"><div class="mediocre-tableEditable-axisCell-labelSizer u-overflow-wrap-break-word invisible mediocre-tableEditable-axisCell--header">A</div><div class="u-flex-display invisible"></div><div class="mediocre-tableEditable-axisCell-label u-verticalCenterItems"><span class="mediocre-tableEditable-axisCell-label-text u-ellipsis-text mediocre-tableEditable-axisCell--header">A</span><div class="u-flex-display"></div></div></div></td><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--col"><div class="mediocre-tableEditable-axisCell-labelWrapper"><div class="mediocre-tableEditable-axisCell-labelSizer u-overflow-wrap-break-word invisible mediocre-tableEditable-axisCell--header">B</div><div class="u-flex-display invisible"></div><div class="mediocre-tableEditable-axisCell-label u-verticalCenterItems"><span class="mediocre-tableEditable-axisCell-label-text u-ellipsis-text mediocre-tableEditable-axisCell--header">B</span><div class="u-flex-display"></div></div></div></td><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--col"><div class="mediocre-tableEditable-axisCell-labelWrapper"><div class="mediocre-tableEditable-axisCell-labelSizer u-overflow-wrap-break-word invisible mediocre-tableEditable-axisCell--header">C</div><div class="u-flex-display invisible"></div><div class="mediocre-tableEditable-axisCell-label u-verticalCenterItems"><span class="mediocre-tableEditable-axisCell-label-text u-ellipsis-text mediocre-tableEditable-axisCell--header">C</span><div class="u-flex-display"></div></div></div></td><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--col"><div class="mediocre-tableEditable-axisCell-labelWrapper"><div class="mediocre-tableEditable-axisCell-labelSizer u-overflow-wrap-break-word invisible mediocre-tableEditable-axisCell--header">D</div><div class="u-flex-display invisible"></div><div class="mediocre-tableEditable-axisCell-label u-verticalCenterItems"><span class="mediocre-tableEditable-axisCell-label-text u-ellipsis-text mediocre-tableEditable-axisCell--header">D</span><div class="u-flex-display"></div></div></div></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div class="mediocre-tableEditable-axisCell-labelWrapper"><div class="mediocre-tableEditable-axisCell-labelSizer u-overflow-wrap-break-word invisible">1</div><div class="u-flex-display invisible"></div><div class="mediocre-tableEditable-axisCell-label u-verticalCenterItems"><span class="mediocre-tableEditable-axisCell-label-text u-ellipsis-text">1</span><div class="u-flex-display"></div></div></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-0-0" data-namespace="table-ns1556"><span>Reagents</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-0-1" data-namespace="table-ns1556"><span>Stock Concentration</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-0-2" data-namespace="table-ns1556"><span>Volume</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-0-3" data-namespace="table-ns1556"><span>Final Concentration </span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div class="mediocre-tableEditable-axisCell-labelWrapper"><div class="mediocre-tableEditable-axisCell-labelSizer u-overflow-wrap-break-word invisible">2</div><div class="u-flex-display invisible"></div><div class="mediocre-tableEditable-axisCell-label u-verticalCenterItems"><span class="mediocre-tableEditable-axisCell-label-text u-ellipsis-text">2</span><div class="u-flex-display"></div></div></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-1-0" data-namespace="table-ns1556"><span>Forward Primer</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-1-1" data-namespace="table-ns1556"><span>100 μM</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-1-2" data-namespace="table-ns1556"><span>15 μL</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-1-3" data-namespace="table-ns1556"><span>1 μM</span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div class="mediocre-tableEditable-axisCell-labelWrapper"><div class="mediocre-tableEditable-axisCell-labelSizer u-overflow-wrap-break-word invisible">3</div><div class="u-flex-display invisible"></div><div class="mediocre-tableEditable-axisCell-label u-verticalCenterItems"><span class="mediocre-tableEditable-axisCell-label-text u-ellipsis-text">3</span><div class="u-flex-display"></div></div></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-2-0" data-namespace="table-ns1556"><span>Reverse Primer</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-2-1" data-namespace="table-ns1556"><span>100 μM</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-2-2" data-namespace="table-ns1556"><span>15 μL</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-2-3" data-namespace="table-ns1556"><span>1 μM</span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div class="mediocre-tableEditable-axisCell-labelWrapper"><div class="mediocre-tableEditable-axisCell-labelSizer u-overflow-wrap-break-word invisible">4</div><div class="u-flex-display invisible"></div><div class="mediocre-tableEditable-axisCell-label u-verticalCenterItems"><span class="mediocre-tableEditable-axisCell-label-text u-ellipsis-text">4</span><div class="u-flex-display"></div></div></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-3-0" data-namespace="table-ns1556"><span>dCTP and dTTP</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-3-1" data-namespace="table-ns1556"><span>20 mM</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-3-2" data-namespace="table-ns1556"><span>75 μL/ea</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-3-3" data-namespace="table-ns1556"><span>1 mM</span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div class="mediocre-tableEditable-axisCell-labelWrapper"><div class="mediocre-tableEditable-axisCell-labelSizer u-overflow-wrap-break-word invisible">5</div><div class="u-flex-display invisible"></div><div class="mediocre-tableEditable-axisCell-label u-verticalCenterItems"><span class="mediocre-tableEditable-axisCell-label-text u-ellipsis-text">5</span><div class="u-flex-display"></div></div></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-4-0" data-namespace="table-ns1556"><span>dATP and dGTP</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-4-1" data-namespace="table-ns1556"><span>20 mM</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-4-2" data-namespace="table-ns1556"><span>15 μL/ea</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-4-3" data-namespace="table-ns1556"><span>0.2 mM</span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div class="mediocre-tableEditable-axisCell-labelWrapper"><div class="mediocre-tableEditable-axisCell-labelSizer u-overflow-wrap-break-word invisible">6</div><div class="u-flex-display invisible"></div><div class="mediocre-tableEditable-axisCell-label u-verticalCenterItems"><span class="mediocre-tableEditable-axisCell-label-text u-ellipsis-text">6</span><div class="u-flex-display"></div></div></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-5-0" data-namespace="table-ns1556"><span>PCR buffer w/ Mg2+</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-5-1" data-namespace="table-ns1556"><span>10X</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-5-2" data-namespace="table-ns1556"><span>150 μL</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-5-3" data-namespace="table-ns1556"><span>1x</span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div class="mediocre-tableEditable-axisCell-labelWrapper"><div class="mediocre-tableEditable-axisCell-labelSizer u-overflow-wrap-break-word invisible">7</div><div class="u-flex-display invisible"></div><div class="mediocre-tableEditable-axisCell-label u-verticalCenterItems"><span class="mediocre-tableEditable-axisCell-label-text u-ellipsis-text">7</span><div class="u-flex-display"></div></div></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-6-0" data-namespace="table-ns1556"><span>MgCl2</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-6-1" data-namespace="table-ns1556"><span>1M</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-6-2" data-namespace="table-ns1556"><span>8 μL</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-6-3" data-namespace="table-ns1556"><span>~5.5 mM</span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div class="mediocre-tableEditable-axisCell-labelWrapper"><div class="mediocre-tableEditable-axisCell-labelSizer u-overflow-wrap-break-word invisible">8</div><div class="u-flex-display invisible"></div><div class="mediocre-tableEditable-axisCell-label u-verticalCenterItems"><span class="mediocre-tableEditable-axisCell-label-text u-ellipsis-text">8</span><div class="u-flex-display"></div></div></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-7-0" data-namespace="table-ns1556"><span>DMDC H2O</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-7-1" data-namespace="table-ns1556"><span></span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-7-2" data-namespace="table-ns1556"><span>1098 μL</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-7-3" data-namespace="table-ns1556"><span></span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div class="mediocre-tableEditable-axisCell-labelWrapper"><div class="mediocre-tableEditable-axisCell-labelSizer u-overflow-wrap-break-word invisible">9</div><div class="u-flex-display invisible"></div><div class="mediocre-tableEditable-axisCell-label u-verticalCenterItems"><span class="mediocre-tableEditable-axisCell-label-text u-ellipsis-text">9</span><div class="u-flex-display"></div></div></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-8-0" data-namespace="table-ns1556"><span>Final Volume </span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-8-1" data-namespace="table-ns1556"><span></span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-8-2" data-namespace="table-ns1556"><span>1491 μL</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-8-3" data-namespace="table-ns1556"><span></span></td></tr></tbody><tfoot></tfoot></table></div></div></div></div><div class="mediocre-tableEditable-titleBarWrapper"><div class="mediocre-tableEditable-titleBar" style="height: 31px; padding-left: 0px;"><div class="mediocre-tableEditable-tableName u-pointer"><div class="u-flex-grow u-overflow-hidden u-verticalCenterItems"><div class="u-ellipsis-text"><span class="u-ellipsis-text">PCR Reagent Mixture</span></div></div><div class="u-flex-none"></div></div><div class="u-flex-none u-tiny-spacing-left table-floater"></div><div class="table-floater-wrapper"></div></div></div></div></div><div class="mediocre-item is-table" data-scroll-id="s910"><div class="mediocre-tableEditable printable-container-scaleForPrint" data-formattable="true" data-index="8" data-subindex="cell"><div><!-- react-empty: 319 --><!-- react-empty: 320 --><!-- react-empty: 321 --></div><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><div class="hiddenFocusEditable editable hiddenFocusEditable--fullTable" contenteditable="true" data-cid="s910-table" data-formattable="true" data-index="8" data-subindex="table"></div><div class="u-position-relative mediocre-tableEditable-scrollAreaWrapper" style="margin-left: 0px; margin-right: 0px;"><div class="mediocre-tableEditable-scrollArea" style="padding-top: 41px;"><div class="mediocre-tableEditable-tableWrapper" style="padding-left: 0px;"><div class="mediocre-tableEditable-border multi-editable" style="left: -6px; top: -37px; bottom: 4px; width: 457px; min-width: 12px;"></div><div class="u-position-relative u-flex-display"><table class="mediocre-tableEditable-table mediocre-tableEditable-table--main" style="width: 445px;"><colgroup><col style="width: 45px;"></col><col data-id="0" style="width: 100px;"></col><col data-id="1" style="width: 100px;"></col><col data-id="2" style="width: 100px;"></col><col data-id="3" style="width: 100px;"></col></colgroup><tbody><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-maximizeButton"><span class="glyphicon glyphicon-arrow-up mediocre-tableEditable-maximizeIcon" icon="arrow-up"></span></td><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--col"><div class="mediocre-tableEditable-axisCell-labelWrapper"><div class="mediocre-tableEditable-axisCell-labelSizer u-overflow-wrap-break-word invisible mediocre-tableEditable-axisCell--header">A</div><div class="u-flex-display invisible"></div><div class="mediocre-tableEditable-axisCell-label u-verticalCenterItems"><span class="mediocre-tableEditable-axisCell-label-text u-ellipsis-text mediocre-tableEditable-axisCell--header">A</span><div class="u-flex-display"></div></div></div></td><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--col"><div class="mediocre-tableEditable-axisCell-labelWrapper"><div class="mediocre-tableEditable-axisCell-labelSizer u-overflow-wrap-break-word invisible mediocre-tableEditable-axisCell--header">B</div><div class="u-flex-display invisible"></div><div class="mediocre-tableEditable-axisCell-label u-verticalCenterItems"><span class="mediocre-tableEditable-axisCell-label-text u-ellipsis-text mediocre-tableEditable-axisCell--header">B</span><div class="u-flex-display"></div></div></div></td><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--col"><div class="mediocre-tableEditable-axisCell-labelWrapper"><div class="mediocre-tableEditable-axisCell-labelSizer u-overflow-wrap-break-word invisible mediocre-tableEditable-axisCell--header">C</div><div class="u-flex-display invisible"></div><div class="mediocre-tableEditable-axisCell-label u-verticalCenterItems"><span class="mediocre-tableEditable-axisCell-label-text u-ellipsis-text mediocre-tableEditable-axisCell--header">C</span><div class="u-flex-display"></div></div></div></td><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--col"><div class="mediocre-tableEditable-axisCell-labelWrapper"><div class="mediocre-tableEditable-axisCell-labelSizer u-overflow-wrap-break-word invisible mediocre-tableEditable-axisCell--header">D</div><div class="u-flex-display invisible"></div><div class="mediocre-tableEditable-axisCell-label u-verticalCenterItems"><span class="mediocre-tableEditable-axisCell-label-text u-ellipsis-text mediocre-tableEditable-axisCell--header">D</span><div class="u-flex-display"></div></div></div></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div class="mediocre-tableEditable-axisCell-labelWrapper"><div class="mediocre-tableEditable-axisCell-labelSizer u-overflow-wrap-break-word invisible">1</div><div class="u-flex-display invisible"></div><div class="mediocre-tableEditable-axisCell-label u-verticalCenterItems"><span class="mediocre-tableEditable-axisCell-label-text u-ellipsis-text">1</span><div class="u-flex-display"></div></div></div></td><td class="cell is-text is-bold is-alignLeft cell-contents" data-cid="table-s910-0-0" data-namespace="table-ns1556"><span>Step</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s910-0-1" data-namespace="table-ns1556"><span>Temperature</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s910-0-2" data-namespace="table-ns1556"><span>Duration</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s910-0-3" data-namespace="table-ns1556"><span></span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div class="mediocre-tableEditable-axisCell-labelWrapper"><div class="mediocre-tableEditable-axisCell-labelSizer u-overflow-wrap-break-word invisible">2</div><div class="u-flex-display invisible"></div><div class="mediocre-tableEditable-axisCell-label u-verticalCenterItems"><span class="mediocre-tableEditable-axisCell-label-text u-ellipsis-text">2</span><div class="u-flex-display"></div></div></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s910-1-0" data-namespace="table-ns1556"><span>Template Denaturation</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s910-1-1" data-namespace="table-ns1556"><span>95 - 98°C</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s910-1-2" data-namespace="table-ns1556"><span>1 min</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s910-1-3" data-namespace="table-ns1556"><span>One time only</span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div class="mediocre-tableEditable-axisCell-labelWrapper"><div class="mediocre-tableEditable-axisCell-labelSizer u-overflow-wrap-break-word invisible">3</div><div class="u-flex-display invisible"></div><div class="mediocre-tableEditable-axisCell-label u-verticalCenterItems"><span class="mediocre-tableEditable-axisCell-label-text u-ellipsis-text">3</span><div class="u-flex-display"></div></div></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s910-2-0" data-namespace="table-ns1556"><span>Oligo Denaturation</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s910-2-1" data-namespace="table-ns1556"><span>94°C</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s910-2-2" data-namespace="table-ns1556"><span>1 min</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s910-2-3" data-namespace="table-ns1556"><span>Repeat next 3 steps x30</span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div class="mediocre-tableEditable-axisCell-labelWrapper"><div class="mediocre-tableEditable-axisCell-labelSizer u-overflow-wrap-break-word invisible">4</div><div class="u-flex-display invisible"></div><div class="mediocre-tableEditable-axisCell-label u-verticalCenterItems"><span class="mediocre-tableEditable-axisCell-label-text u-ellipsis-text">4</span><div class="u-flex-display"></div></div></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s910-3-0" data-namespace="table-ns1556"><span>Annealing</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s910-3-1" data-namespace="table-ns1556"><span>58°C</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s910-3-2" data-namespace="table-ns1556"><span>1 min</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s910-3-3" data-namespace="table-ns1556"><span>This may change depending on primers </span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div class="mediocre-tableEditable-axisCell-labelWrapper"><div class="mediocre-tableEditable-axisCell-labelSizer u-overflow-wrap-break-word invisible">5</div><div class="u-flex-display invisible"></div><div class="mediocre-tableEditable-axisCell-label u-verticalCenterItems"><span class="mediocre-tableEditable-axisCell-label-text u-ellipsis-text">5</span><div class="u-flex-display"></div></div></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s910-4-0" data-namespace="table-ns1556"><span>Extension</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s910-4-1" data-namespace="table-ns1556"><span>72°C</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s910-4-2" data-namespace="table-ns1556"><span>2 min</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s910-4-3" data-namespace="table-ns1556"><span></span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div class="mediocre-tableEditable-axisCell-labelWrapper"><div class="mediocre-tableEditable-axisCell-labelSizer u-overflow-wrap-break-word invisible">6</div><div class="u-flex-display invisible"></div><div class="mediocre-tableEditable-axisCell-label u-verticalCenterItems"><span class="mediocre-tableEditable-axisCell-label-text u-ellipsis-text">6</span><div class="u-flex-display"></div></div></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s910-5-0" data-namespace="table-ns1556"><span>Final Elongation</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s910-5-1" data-namespace="table-ns1556"><span>72°C</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s910-5-2" data-namespace="table-ns1556"><span>5 min</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s910-5-3" data-namespace="table-ns1556"><span></span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div class="mediocre-tableEditable-axisCell-labelWrapper"><div class="mediocre-tableEditable-axisCell-labelSizer u-overflow-wrap-break-word invisible">7</div><div class="u-flex-display invisible"></div><div class="mediocre-tableEditable-axisCell-label u-verticalCenterItems"><span class="mediocre-tableEditable-axisCell-label-text u-ellipsis-text">7</span><div class="u-flex-display"></div></div></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s910-6-0" data-namespace="table-ns1556"><span>Product Storage</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s910-6-1" data-namespace="table-ns1556"><span>4°C</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s910-6-2" data-namespace="table-ns1556"><span>forever</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s910-6-3" data-namespace="table-ns1556"><span>Repeat steps 3-5 for 30 cycles </span></td></tr></tbody><tfoot></tfoot></table></div></div></div></div><div class="mediocre-tableEditable-titleBarWrapper"><div class="mediocre-tableEditable-titleBar" style="height: 31px; padding-left: 0px;"><div class="mediocre-tableEditable-tableName u-pointer"><div class="u-flex-grow u-overflow-hidden u-verticalCenterItems"><div class="u-ellipsis-text"><span class="u-ellipsis-text">Table1</span></div></div><div class="u-flex-none"></div></div><div class="u-flex-none u-tiny-spacing-left table-floater"></div><div class="table-floater-wrapper"></div></div></div></div></div><div class="mediocre-item is-step" data-scroll-id="s911"><ol class="steps"><li value="7"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s911" data-index="9">Once the PCR program has reached the annealing temperature, add 1 <span style="color:#333333"><span style="background-color:#FFFFFF">μL of freshly prepared MnCl2 solution and 1 </span></span><span style="color:#333333"><span style="background-color:#FFFFFF">μL of <i>Taq </i>DNA polymerase to the PCR reaction tube. </span></span></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s911" data-index="9" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s912"><ol class="steps"><li value="8"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s912" data-index="10">Perform 4 cycles of PCR amplification using the hot start procedure (see Note 3). </div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s912" data-index="10" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s913"><ol class="steps"><li value="9"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s913" data-index="11">Remove the PCR tube from the thermocycler</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s913" data-index="11" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s914"><ol class="steps"><li value="10"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s914" data-index="12">Place the tube on ice. </div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s914" data-index="12" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s915"><ol class="steps"><li value="11"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s915" data-index="13">Transfer 10 <span style="color:#333333"><span style="background-color:#FFFFFF">μL of the PCR reaction from tube 1 to tube 2. </span></span></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s915" data-index="13" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s916"><ol class="steps"><li value="12"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s916" data-index="14">Place tube 2 in the thermocycler and start the PCR program. </div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s916" data-index="14" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s917"><ol class="steps"><li value="13"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s917" data-index="15">Once the PCR program has reached the annealing temperature, add 1 <span style="color:#333333"><span style="background-color:#FFFFFF">μL of freshly prepared MnCl2 and 1 </span></span><span style="color:#333333"><span style="background-color:#FFFFFF">μL of <i>Taq </i>DNA polymerase to the PCR reaction tube. </span></span></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s917" data-index="15" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s918"><ol class="steps"><li value="14"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s918" data-index="16">Perform 4 cycles of PCR amplification using the host start procedure. </div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s918" data-index="16" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s919"><ol class="steps"><li value="15"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s919" data-index="17">Remove the PCR tube from the thermocycler</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s919" data-index="17" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s920"><ol class="steps"><li value="16"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s920" data-index="18">Place the tube on ice. </div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s920" data-index="18" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s921"><ol class="steps"><li value="17"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s921" data-index="19">R<span style="color:#333333"><span style="background-color:#FFFFFF">epeat steps 11 through 16 using tubes 3 through 16 to create a mutagenic library by serial dilution amplification (image down below)</span></span></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s921" data-index="19" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s922"><ol class="steps"><li value="18"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s922" data-index="20"> Verify the quality of each PCR reaction by agarose gel electrophoresis. </div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s922" data-index="20" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s923"><ol class="steps"><li value="19"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s923" data-index="21"><span style="color:#333333"><span style="background-color:#FFFFFF">Combine 50 μL of each PCR reaction into a single tube and store on ice; this is your DNA library</span></span></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s923" data-index="21" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s924"><ol class="steps"><li value="20"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s924" data-index="22"><span style="color:#333333"><span style="background-color:#FFFFFF">Purify the DNA library by agarose gel electrophoresis</span></span></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s924" data-index="22" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s925"><ol class="steps"><li value="21"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s925" data-index="23"><span style="color:#333333"><span style="background-color:#FFFFFF">Recover the DNA library using a gel purification kit.</span></span></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s925" data-index="23" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s926"><ol class="steps"><li value="22"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s926" data-index="24"><span style="color:#333333"><span style="background-color:#FFFFFF">Quantify the DNA library by agarose gel electrophoresis</span></span></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s926" data-index="24" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-header" data-scroll-id="s927"><ul class="headers"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s927" data-index="25">NOTES</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s927" data-index="25" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ul></div><div class="mediocre-item is-step" data-scroll-id="s928"><ol class="steps"><li value="23"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s928" data-index="26"><span style="color:#8E2555"><span style="background-color:#FFFFFF">Primer should be complemetary and have similar melting techniques: </span></span><a href="http://bioweb.uwlax.edu/GenWeb/Molecular/seq_anal/primer_design/primer_design.htm" rel="nofollow" target="_blank"><span style="color:#8E2555"><span style="background-color:#FFFFFF">http://bioweb.uwlax.edu/GenWeb/Molecular/seq_anal/primer_design/primer_design.htm</span></span></a></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s928" data-index="26" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s929"><ol class="steps"><li value="24"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s929" data-index="27"><span style="color:#333333"><span style="background-color:#FFFFFF">Optimal PCR conditions should be determined prior to the use of this mutagenic PCR protocol. The doubling efficiency for the normal PCR reaction should be ∼1.7–1.9 per PCR cycle </span></span><span style="color:#333333"><span style="background-color:#FFFFFF"> and can be analyzed by running the PCR product after each cycle on an agarose gel</span></span></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s929" data-index="27" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s930"><ol class="steps"><li value="25"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s930" data-index="28"><i><span style="color:#333333"><span style="background-color:#FFFFFF">Taq</span></span></i><span style="color:#333333"><span style="background-color:#FFFFFF"> DNA polymerase should be stored at −20°C and kept on ice or in a frozen metal block when taken out of the freezer. The hot start procedure refers to adding the </span></span><i><span style="color:#333333"><span style="background-color:#FFFFFF">Taq</span></span></i><span style="color:#333333"><span style="background-color:#FFFFFF"> DNA polymerase to the PCR reaction mixture after the mixture has reached the annealing temperature. Either standard or “hot start” </span></span><i><span style="color:#333333"><span style="background-color:#FFFFFF">Taq</span></span></i><span style="color:#333333"><span style="background-color:#FFFFFF"> DNA polymerase will work in the described method.</span></span></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s930" data-index="28" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s931"><ol class="steps"><li value="26"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s931" data-index="29"><span style="color:#333333"><span style="background-color:#FFFFFF">It is important to prepare a fresh MnCl</span></span><sub><span style="color:#333333"><span style="background-color:#FFFFFF">2</span></span></sub><span style="color:#333333"><span style="background-color:#FFFFFF"> solution for the error-prone PCR experiment. This solution should be stored on ice and combined with the PCR reaction mixture at the start of each serial dilution.</span></span></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s931" data-index="29" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s932"><ol class="steps"><li value="27"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s932" data-index="30"><span style="color:#333333"><span style="background-color:#FFFFFF">It is important to prepare a fresh MnCl</span></span><sub><span style="color:#333333"><span style="background-color:#FFFFFF">2</span></span></sub><span style="color:#333333"><span style="background-color:#FFFFFF"> solution for the error-prone PCR experiment. This solution should be stored on ice and combined with the PCR reaction mixture at the start of each serial dilution.</span></span></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s932" data-index="30" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s933"><ol class="steps"><li value="28"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s933" data-index="31"><span style="color:#333333"><span style="background-color:#FFFFFF">The method described here was optimized for a 400-nucleotide gene. We highly recommend that experimenters read ref. </span></span><a href="https://link.springer.com/protocol/10.1007/978-1-60761-652-8_7#CR7_7" rel="nofollow" target="_blank"><u><span style="color:#333333"><span style="color:#8E2555"><span style="background-color:#FFFFFF">7</span></span></span></u></a><span style="color:#333333"><span style="background-color:#FFFFFF"> for information on the predicted average number of doublings and mutations expected as a function of template length</span></span></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s933" data-index="31" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s934"><ol class="steps"><li value="29"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s934" data-index="32"><span style="color:#333333"><span style="background-color:#FFFFFF">Agarose gel electrophoresis should be performed by standard methods. A DNA ladder with an appropriate base pair range should be used to identify and estimate the quantity of the PCR product.</span></span></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s934" data-index="32" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s935"><ol class="steps"><li value="30"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s935" data-index="33"><span style="color:#333333"><span style="background-color:#FFFFFF">It is recommended to save a portion of the PCR product from each tube for further analysis or amplification.</span></span></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s935" data-index="33" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s936"><ol class="steps"><li value="31"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s936" data-index="34"><span style="color:#333333"><span style="background-color:#FFFFFF">The concentration of the DNA product can be determined by comparing the intensity of the product bands to the intensity of the bands from the DNA quantification ladder from commercial sources.</span></span></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s936" data-index="34" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-header" data-scroll-id="s937"><ul class="headers"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s937" data-index="35">Transformation of Libraries </div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s937" data-index="35" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ul></div><div class="mediocre-item is-step" data-scroll-id="s938"><ol class="steps"><li value="32"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s938" data-index="36">GIbson Assembely can be performed if the primers of the linearized vector are complimentery to the error-prone PCR product.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s938" data-index="36" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-image" data-scroll-id="s939"><div class="image-editable"><form class="add-image-form"><input accept="image/*" class="image-input hidden" type="file"/><div class="upload-wrapper multi-editable"><img class="img" src="assets/proto-image-6deQnbrh4mWOszXlP3WfJEmOMdNtdLEdCtFAX5FS-Screen Shot 2018-05-17 at 2.19.41 PM.png"/></div><div class="progress-bar" style="width: 0%;"></div></form></div></div></div></div></div></div></div></div></div></div></div><!-- react-empty: 721 --></div></div></div>
+</div></div><h2>Materials</h2><div class="materials-editor" data-editor="materials"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><ul class="materials"><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m888" data-index="0"></div><ul class="components"><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c889" data-index="0,0">DNA primers for PCR amplification</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c890" data-index="0,1">DNA template</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c891" data-index="0,2">Four separate solutions of 20 mM dATP, TTP, dCTP, and dGTP</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c892" data-index="0,3"><i>Taq </i>DNA polymerase, stored at -20 C</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c893" data-index="0,4">10 X PCR buffer: 100 mM Tris-HCl, pH 8.3, 15 mM MgCl2, and 500 mM KCl</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c894" data-index="0,5">1 MgCl2</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c895" data-index="0,6">50 mM MnCl2</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c896" data-index="0,7">Agarose</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c897" data-index="0,8">10 X TBE buffer: 1 M Tris base, 1 M boric acid, and 20 mM EDTA, pH 8.0</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c898" data-index="0,9">10 mg/mL ethidium bromide </div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c899" data-index="0,10">Loading buffer: 10 mM Tris-HCl, pH 7.6, 0.15 % orange G, 0.03% xylene cyanol FF, 60% glycerol, 60 mM EDTA</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c900" data-index="0,11">DNA mass ladder for quantification</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c901" data-index="0,12">Gel purification kit </div></li></ul></li></ul></div><h2>Procedure</h2><div class="stepsEditor mediocre"><div class="editor" data-editor="steps"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><div class="mediocre-item is-header" data-scroll-id="s902"><ul class="headers"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s902" data-index="0"></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s902" data-index="0" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ul></div><div class="mediocre-item is-step" data-scroll-id="s903"><ol class="steps"><li value="1"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s903" data-index="1">Prepare a stock solution of the DNA template (~50-100 ng/<span style="color:#333333"><span style="background-color:#FFFFFF">μL) in water. </span></span></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s903" data-index="1" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s904"><ol class="steps"><li value="2"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s904" data-index="2">Label 16 thin-walled PCR tubes as "reactions 1 through 16".</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s904" data-index="2" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s905"><ol class="steps"><li value="3"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s905" data-index="3">Combine the reagents listed in Table 1 in a conical vial and label it "PCR reaction mixture"</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s905" data-index="3" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s906"><ol class="steps"><li value="4"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s906" data-index="4">Dispense the PCR reaction mixture from <b>step 3 </b>into tubes 1 through 16 by 96 <span style="color:#344043"><span style="background-color:#FFFFFF">μL to tube 1 and add 88 </span></span><span style="color:#344043"><span style="background-color:#FFFFFF">μL to tubes 2-16. </span></span></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s906" data-index="4" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s907"><ol class="steps"><li value="5"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s907" data-index="5">Add 2 <span style="color:#344043"><span style="background-color:#FFFFFF">μL of the DNA template to tube 1.</span></span></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s907" data-index="5" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s908"><ol class="steps"><li value="6"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s908" data-index="6">Place tube 1 in the thermocycler and start the PCR program (Table 2).</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s908" data-index="6" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-table" data-scroll-id="s909"><div class="mediocre-tableEditable printable-container-scaleForPrint" data-formattable="true" data-index="7" data-subindex="cell"><div><!-- react-empty: 113 --><!-- react-empty: 114 --><!-- react-empty: 115 --></div><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><div class="hiddenFocusEditable editable hiddenFocusEditable--fullTable" contenteditable="true" data-cid="s909-table" data-formattable="true" data-index="7" data-subindex="table"></div><div class="u-position-relative mediocre-tableEditable-scrollAreaWrapper" style="margin-left: 0px; margin-right: 0px;"><div class="mediocre-tableEditable-scrollArea" style="padding-top: 41px;"><div class="mediocre-tableEditable-tableWrapper" style="padding-left: 0px;"><div class="mediocre-tableEditable-border multi-editable" style="left: -6px; top: -37px; bottom: 4px; width: 457px; min-width: 12px;"></div><div class="u-position-relative u-flex-display"><table class="mediocre-tableEditable-table mediocre-tableEditable-table--main" style="width: 445px;"><colgroup><col style="width: 45px;"></col><col data-id="0" style="width: 100px;"></col><col data-id="1" style="width: 100px;"></col><col data-id="2" style="width: 100px;"></col><col data-id="3" style="width: 100px;"></col></colgroup><tbody><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-maximizeButton"><span class="glyphicon glyphicon-arrow-up mediocre-tableEditable-maximizeIcon" icon="arrow-up"></span></td><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--col"><div class="mediocre-tableEditable-axisCell-labelWrapper"><div class="mediocre-tableEditable-axisCell-labelSizer u-overflow-wrap-break-word invisible mediocre-tableEditable-axisCell--header">A</div><div class="u-flex-display invisible"></div><div class="mediocre-tableEditable-axisCell-label u-verticalCenterItems"><span class="mediocre-tableEditable-axisCell-label-text u-ellipsis-text mediocre-tableEditable-axisCell--header">A</span><div class="u-flex-display"></div></div></div></td><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--col"><div class="mediocre-tableEditable-axisCell-labelWrapper"><div class="mediocre-tableEditable-axisCell-labelSizer u-overflow-wrap-break-word invisible mediocre-tableEditable-axisCell--header">B</div><div class="u-flex-display invisible"></div><div class="mediocre-tableEditable-axisCell-label u-verticalCenterItems"><span class="mediocre-tableEditable-axisCell-label-text u-ellipsis-text mediocre-tableEditable-axisCell--header">B</span><div class="u-flex-display"></div></div></div></td><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--col"><div class="mediocre-tableEditable-axisCell-labelWrapper"><div class="mediocre-tableEditable-axisCell-labelSizer u-overflow-wrap-break-word invisible mediocre-tableEditable-axisCell--header">C</div><div class="u-flex-display invisible"></div><div class="mediocre-tableEditable-axisCell-label u-verticalCenterItems"><span class="mediocre-tableEditable-axisCell-label-text u-ellipsis-text mediocre-tableEditable-axisCell--header">C</span><div class="u-flex-display"></div></div></div></td><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--col"><div class="mediocre-tableEditable-axisCell-labelWrapper"><div class="mediocre-tableEditable-axisCell-labelSizer u-overflow-wrap-break-word invisible mediocre-tableEditable-axisCell--header">D</div><div class="u-flex-display invisible"></div><div class="mediocre-tableEditable-axisCell-label u-verticalCenterItems"><span class="mediocre-tableEditable-axisCell-label-text u-ellipsis-text mediocre-tableEditable-axisCell--header">D</span><div class="u-flex-display"></div></div></div></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div class="mediocre-tableEditable-axisCell-labelWrapper"><div class="mediocre-tableEditable-axisCell-labelSizer u-overflow-wrap-break-word invisible">1</div><div class="u-flex-display invisible"></div><div class="mediocre-tableEditable-axisCell-label u-verticalCenterItems"><span class="mediocre-tableEditable-axisCell-label-text u-ellipsis-text">1</span><div class="u-flex-display"></div></div></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-0-0" data-namespace="table-ns1556"><span>Reagents</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-0-1" data-namespace="table-ns1556"><span>Stock Concentration</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-0-2" data-namespace="table-ns1556"><span>Volume</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-0-3" data-namespace="table-ns1556"><span>Final Concentration </span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div class="mediocre-tableEditable-axisCell-labelWrapper"><div class="mediocre-tableEditable-axisCell-labelSizer u-overflow-wrap-break-word invisible">2</div><div class="u-flex-display invisible"></div><div class="mediocre-tableEditable-axisCell-label u-verticalCenterItems"><span class="mediocre-tableEditable-axisCell-label-text u-ellipsis-text">2</span><div class="u-flex-display"></div></div></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-1-0" data-namespace="table-ns1556"><span>Forward Primer</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-1-1" data-namespace="table-ns1556"><span>100 μM</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-1-2" data-namespace="table-ns1556"><span>15 μL</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-1-3" data-namespace="table-ns1556"><span>1 μM</span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div class="mediocre-tableEditable-axisCell-labelWrapper"><div class="mediocre-tableEditable-axisCell-labelSizer u-overflow-wrap-break-word invisible">3</div><div class="u-flex-display invisible"></div><div class="mediocre-tableEditable-axisCell-label u-verticalCenterItems"><span class="mediocre-tableEditable-axisCell-label-text u-ellipsis-text">3</span><div class="u-flex-display"></div></div></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-2-0" data-namespace="table-ns1556"><span>Reverse Primer</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-2-1" data-namespace="table-ns1556"><span>100 μM</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-2-2" data-namespace="table-ns1556"><span>15 μL</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-2-3" data-namespace="table-ns1556"><span>1 μM</span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div class="mediocre-tableEditable-axisCell-labelWrapper"><div class="mediocre-tableEditable-axisCell-labelSizer u-overflow-wrap-break-word invisible">4</div><div class="u-flex-display invisible"></div><div class="mediocre-tableEditable-axisCell-label u-verticalCenterItems"><span class="mediocre-tableEditable-axisCell-label-text u-ellipsis-text">4</span><div class="u-flex-display"></div></div></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-3-0" data-namespace="table-ns1556"><span>dCTP and dTTP</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-3-1" data-namespace="table-ns1556"><span>20 mM</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-3-2" data-namespace="table-ns1556"><span>75 μL/ea</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-3-3" data-namespace="table-ns1556"><span>1 mM</span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div class="mediocre-tableEditable-axisCell-labelWrapper"><div class="mediocre-tableEditable-axisCell-labelSizer u-overflow-wrap-break-word invisible">5</div><div class="u-flex-display invisible"></div><div class="mediocre-tableEditable-axisCell-label u-verticalCenterItems"><span class="mediocre-tableEditable-axisCell-label-text u-ellipsis-text">5</span><div class="u-flex-display"></div></div></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-4-0" data-namespace="table-ns1556"><span>dATP and dGTP</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-4-1" data-namespace="table-ns1556"><span>20 mM</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-4-2" data-namespace="table-ns1556"><span>15 μL/ea</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-4-3" data-namespace="table-ns1556"><span>0.2 mM</span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div class="mediocre-tableEditable-axisCell-labelWrapper"><div class="mediocre-tableEditable-axisCell-labelSizer u-overflow-wrap-break-word invisible">6</div><div class="u-flex-display invisible"></div><div class="mediocre-tableEditable-axisCell-label u-verticalCenterItems"><span class="mediocre-tableEditable-axisCell-label-text u-ellipsis-text">6</span><div class="u-flex-display"></div></div></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-5-0" data-namespace="table-ns1556"><span>PCR buffer w/ Mg2+</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-5-1" data-namespace="table-ns1556"><span>10X</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-5-2" data-namespace="table-ns1556"><span>150 μL</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-5-3" data-namespace="table-ns1556"><span>1x</span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div class="mediocre-tableEditable-axisCell-labelWrapper"><div class="mediocre-tableEditable-axisCell-labelSizer u-overflow-wrap-break-word invisible">7</div><div class="u-flex-display invisible"></div><div class="mediocre-tableEditable-axisCell-label u-verticalCenterItems"><span class="mediocre-tableEditable-axisCell-label-text u-ellipsis-text">7</span><div class="u-flex-display"></div></div></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-6-0" data-namespace="table-ns1556"><span>MgCl2</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-6-1" data-namespace="table-ns1556"><span>1M</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-6-2" data-namespace="table-ns1556"><span>8 μL</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-6-3" data-namespace="table-ns1556"><span>~5.5 mM</span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div class="mediocre-tableEditable-axisCell-labelWrapper"><div class="mediocre-tableEditable-axisCell-labelSizer u-overflow-wrap-break-word invisible">8</div><div class="u-flex-display invisible"></div><div class="mediocre-tableEditable-axisCell-label u-verticalCenterItems"><span class="mediocre-tableEditable-axisCell-label-text u-ellipsis-text">8</span><div class="u-flex-display"></div></div></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-7-0" data-namespace="table-ns1556"><span>DMDC H2O</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-7-1" data-namespace="table-ns1556"><span></span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-7-2" data-namespace="table-ns1556"><span>1098 μL</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-7-3" data-namespace="table-ns1556"><span></span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div class="mediocre-tableEditable-axisCell-labelWrapper"><div class="mediocre-tableEditable-axisCell-labelSizer u-overflow-wrap-break-word invisible">9</div><div class="u-flex-display invisible"></div><div class="mediocre-tableEditable-axisCell-label u-verticalCenterItems"><span class="mediocre-tableEditable-axisCell-label-text u-ellipsis-text">9</span><div class="u-flex-display"></div></div></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-8-0" data-namespace="table-ns1556"><span>Final Volume </span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-8-1" data-namespace="table-ns1556"><span></span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-8-2" data-namespace="table-ns1556"><span>1491 μL</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s909-8-3" data-namespace="table-ns1556"><span></span></td></tr></tbody><tfoot></tfoot></table></div></div></div></div><div class="mediocre-tableEditable-titleBarWrapper"><div class="mediocre-tableEditable-titleBar" style="height: 31px; padding-left: 0px;"><div class="mediocre-tableEditable-tableName u-pointer"><div class="u-flex-grow u-overflow-hidden u-verticalCenterItems"><div class="u-ellipsis-text"><span class="u-ellipsis-text">PCR Reagent Mixture</span></div></div><div class="u-flex-none"></div></div><div class="u-flex-none u-tiny-spacing-left table-floater"></div><div class="table-floater-wrapper"></div></div></div></div></div><div class="mediocre-item is-table" data-scroll-id="s910"><div class="mediocre-tableEditable printable-container-scaleForPrint" data-formattable="true" data-index="8" data-subindex="cell"><div><!-- react-empty: 319 --><!-- react-empty: 320 --><!-- react-empty: 321 --></div><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><div class="hiddenFocusEditable editable hiddenFocusEditable--fullTable" contenteditable="true" data-cid="s910-table" data-formattable="true" data-index="8" data-subindex="table"></div><div class="u-position-relative mediocre-tableEditable-scrollAreaWrapper" style="margin-left: 0px; margin-right: 0px;"><div class="mediocre-tableEditable-scrollArea" style="padding-top: 41px;"><div class="mediocre-tableEditable-tableWrapper" style="padding-left: 0px;"><div class="mediocre-tableEditable-border multi-editable" style="left: -6px; top: -37px; bottom: 4px; width: 457px; min-width: 12px;"></div><div class="u-position-relative u-flex-display"><table class="mediocre-tableEditable-table mediocre-tableEditable-table--main" style="width: 445px;"><colgroup><col style="width: 45px;"></col><col data-id="0" style="width: 100px;"></col><col data-id="1" style="width: 100px;"></col><col data-id="2" style="width: 100px;"></col><col data-id="3" style="width: 100px;"></col></colgroup><tbody><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-maximizeButton"><span class="glyphicon glyphicon-arrow-up mediocre-tableEditable-maximizeIcon" icon="arrow-up"></span></td><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--col"><div class="mediocre-tableEditable-axisCell-labelWrapper"><div class="mediocre-tableEditable-axisCell-labelSizer u-overflow-wrap-break-word invisible mediocre-tableEditable-axisCell--header">A</div><div class="u-flex-display invisible"></div><div class="mediocre-tableEditable-axisCell-label u-verticalCenterItems"><span class="mediocre-tableEditable-axisCell-label-text u-ellipsis-text mediocre-tableEditable-axisCell--header">A</span><div class="u-flex-display"></div></div></div></td><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--col"><div class="mediocre-tableEditable-axisCell-labelWrapper"><div class="mediocre-tableEditable-axisCell-labelSizer u-overflow-wrap-break-word invisible mediocre-tableEditable-axisCell--header">B</div><div class="u-flex-display invisible"></div><div class="mediocre-tableEditable-axisCell-label u-verticalCenterItems"><span class="mediocre-tableEditable-axisCell-label-text u-ellipsis-text mediocre-tableEditable-axisCell--header">B</span><div class="u-flex-display"></div></div></div></td><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--col"><div class="mediocre-tableEditable-axisCell-labelWrapper"><div class="mediocre-tableEditable-axisCell-labelSizer u-overflow-wrap-break-word invisible mediocre-tableEditable-axisCell--header">C</div><div class="u-flex-display invisible"></div><div class="mediocre-tableEditable-axisCell-label u-verticalCenterItems"><span class="mediocre-tableEditable-axisCell-label-text u-ellipsis-text mediocre-tableEditable-axisCell--header">C</span><div class="u-flex-display"></div></div></div></td><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--col"><div class="mediocre-tableEditable-axisCell-labelWrapper"><div class="mediocre-tableEditable-axisCell-labelSizer u-overflow-wrap-break-word invisible mediocre-tableEditable-axisCell--header">D</div><div class="u-flex-display invisible"></div><div class="mediocre-tableEditable-axisCell-label u-verticalCenterItems"><span class="mediocre-tableEditable-axisCell-label-text u-ellipsis-text mediocre-tableEditable-axisCell--header">D</span><div class="u-flex-display"></div></div></div></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div class="mediocre-tableEditable-axisCell-labelWrapper"><div class="mediocre-tableEditable-axisCell-labelSizer u-overflow-wrap-break-word invisible">1</div><div class="u-flex-display invisible"></div><div class="mediocre-tableEditable-axisCell-label u-verticalCenterItems"><span class="mediocre-tableEditable-axisCell-label-text u-ellipsis-text">1</span><div class="u-flex-display"></div></div></div></td><td class="cell is-text is-bold is-alignLeft cell-contents" data-cid="table-s910-0-0" data-namespace="table-ns1556"><span>Step</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s910-0-1" data-namespace="table-ns1556"><span>Temperature</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s910-0-2" data-namespace="table-ns1556"><span>Duration</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s910-0-3" data-namespace="table-ns1556"><span></span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div class="mediocre-tableEditable-axisCell-labelWrapper"><div class="mediocre-tableEditable-axisCell-labelSizer u-overflow-wrap-break-word invisible">2</div><div class="u-flex-display invisible"></div><div class="mediocre-tableEditable-axisCell-label u-verticalCenterItems"><span class="mediocre-tableEditable-axisCell-label-text u-ellipsis-text">2</span><div class="u-flex-display"></div></div></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s910-1-0" data-namespace="table-ns1556"><span>Template Denaturation</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s910-1-1" data-namespace="table-ns1556"><span>95 - 98°C</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s910-1-2" data-namespace="table-ns1556"><span>1 min</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s910-1-3" data-namespace="table-ns1556"><span>One time only</span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div class="mediocre-tableEditable-axisCell-labelWrapper"><div class="mediocre-tableEditable-axisCell-labelSizer u-overflow-wrap-break-word invisible">3</div><div class="u-flex-display invisible"></div><div class="mediocre-tableEditable-axisCell-label u-verticalCenterItems"><span class="mediocre-tableEditable-axisCell-label-text u-ellipsis-text">3</span><div class="u-flex-display"></div></div></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s910-2-0" data-namespace="table-ns1556"><span>Oligo Denaturation</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s910-2-1" data-namespace="table-ns1556"><span>94°C</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s910-2-2" data-namespace="table-ns1556"><span>1 min</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s910-2-3" data-namespace="table-ns1556"><span>Repeat next 3 steps x30</span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div class="mediocre-tableEditable-axisCell-labelWrapper"><div class="mediocre-tableEditable-axisCell-labelSizer u-overflow-wrap-break-word invisible">4</div><div class="u-flex-display invisible"></div><div class="mediocre-tableEditable-axisCell-label u-verticalCenterItems"><span class="mediocre-tableEditable-axisCell-label-text u-ellipsis-text">4</span><div class="u-flex-display"></div></div></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s910-3-0" data-namespace="table-ns1556"><span>Annealing</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s910-3-1" data-namespace="table-ns1556"><span>58°C</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s910-3-2" data-namespace="table-ns1556"><span>1 min</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s910-3-3" data-namespace="table-ns1556"><span>This may change depending on primers </span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div class="mediocre-tableEditable-axisCell-labelWrapper"><div class="mediocre-tableEditable-axisCell-labelSizer u-overflow-wrap-break-word invisible">5</div><div class="u-flex-display invisible"></div><div class="mediocre-tableEditable-axisCell-label u-verticalCenterItems"><span class="mediocre-tableEditable-axisCell-label-text u-ellipsis-text">5</span><div class="u-flex-display"></div></div></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s910-4-0" data-namespace="table-ns1556"><span>Extension</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s910-4-1" data-namespace="table-ns1556"><span>72°C</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s910-4-2" data-namespace="table-ns1556"><span>2 min</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s910-4-3" data-namespace="table-ns1556"><span></span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div class="mediocre-tableEditable-axisCell-labelWrapper"><div class="mediocre-tableEditable-axisCell-labelSizer u-overflow-wrap-break-word invisible">6</div><div class="u-flex-display invisible"></div><div class="mediocre-tableEditable-axisCell-label u-verticalCenterItems"><span class="mediocre-tableEditable-axisCell-label-text u-ellipsis-text">6</span><div class="u-flex-display"></div></div></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s910-5-0" data-namespace="table-ns1556"><span>Final Elongation</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s910-5-1" data-namespace="table-ns1556"><span>72°C</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s910-5-2" data-namespace="table-ns1556"><span>5 min</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s910-5-3" data-namespace="table-ns1556"><span></span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div class="mediocre-tableEditable-axisCell-labelWrapper"><div class="mediocre-tableEditable-axisCell-labelSizer u-overflow-wrap-break-word invisible">7</div><div class="u-flex-display invisible"></div><div class="mediocre-tableEditable-axisCell-label u-verticalCenterItems"><span class="mediocre-tableEditable-axisCell-label-text u-ellipsis-text">7</span><div class="u-flex-display"></div></div></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s910-6-0" data-namespace="table-ns1556"><span>Product Storage</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s910-6-1" data-namespace="table-ns1556"><span>4°C</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s910-6-2" data-namespace="table-ns1556"><span>forever</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s910-6-3" data-namespace="table-ns1556"><span>Repeat steps 3-5 for 30 cycles </span></td></tr></tbody><tfoot></tfoot></table></div></div></div></div><div class="mediocre-tableEditable-titleBarWrapper"><div class="mediocre-tableEditable-titleBar" style="height: 31px; padding-left: 0px;"><div class="mediocre-tableEditable-tableName u-pointer"><div class="u-flex-grow u-overflow-hidden u-verticalCenterItems"><div class="u-ellipsis-text"><span class="u-ellipsis-text">Table1</span></div></div><div class="u-flex-none"></div></div><div class="u-flex-none u-tiny-spacing-left table-floater"></div><div class="table-floater-wrapper"></div></div></div></div></div><div class="mediocre-item is-step" data-scroll-id="s911"><ol class="steps"><li value="7"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s911" data-index="9">Once the PCR program has reached the annealing temperature, add 1 <span style="color:#333333"><span style="background-color:#FFFFFF">μL of freshly prepared MnCl2 solution and 1 </span></span><span style="color:#333333"><span style="background-color:#FFFFFF">μL of <i>Taq </i>DNA polymerase to the PCR reaction tube. </span></span></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s911" data-index="9" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s912"><ol class="steps"><li value="8"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s912" data-index="10">Perform 4 cycles of PCR amplification using the hot start procedure (see Note 3). </div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s912" data-index="10" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s913"><ol class="steps"><li value="9"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s913" data-index="11">Remove the PCR tube from the thermocycler</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s913" data-index="11" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s914"><ol class="steps"><li value="10"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s914" data-index="12">Place the tube on ice. </div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s914" data-index="12" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s915"><ol class="steps"><li value="11"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s915" data-index="13">Transfer 10 <span style="color:#333333"><span style="background-color:#FFFFFF">μL of the PCR reaction from tube 1 to tube 2. </span></span></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s915" data-index="13" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s916"><ol class="steps"><li value="12"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s916" data-index="14">Place tube 2 in the thermocycler and start the PCR program. </div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s916" data-index="14" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s917"><ol class="steps"><li value="13"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s917" data-index="15">Once the PCR program has reached the annealing temperature, add 1 <span style="color:#333333"><span style="background-color:#FFFFFF">μL of freshly prepared MnCl2 and 1 </span></span><span style="color:#333333"><span style="background-color:#FFFFFF">μL of <i>Taq </i>DNA polymerase to the PCR reaction tube. </span></span></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s917" data-index="15" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s918"><ol class="steps"><li value="14"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s918" data-index="16">Perform 4 cycles of PCR amplification using the host start procedure. </div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s918" data-index="16" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s919"><ol class="steps"><li value="15"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s919" data-index="17">Remove the PCR tube from the thermocycler</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s919" data-index="17" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s920"><ol class="steps"><li value="16"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s920" data-index="18">Place the tube on ice. </div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s920" data-index="18" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s921"><ol class="steps"><li value="17"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s921" data-index="19">R<span style="color:#333333"><span style="background-color:#FFFFFF">epeat steps 11 through 16 using tubes 3 through 16 to create a mutagenic library by serial dilution amplification (image down below)</span></span></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s921" data-index="19" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s922"><ol class="steps"><li value="18"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s922" data-index="20"> Verify the quality of each PCR reaction by agarose gel electrophoresis. </div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s922" data-index="20" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s923"><ol class="steps"><li value="19"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s923" data-index="21"><span style="color:#333333"><span style="background-color:#FFFFFF">Combine 50 μL of each PCR reaction into a single tube and store on ice; this is your DNA library</span></span></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s923" data-index="21" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s924"><ol class="steps"><li value="20"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s924" data-index="22"><span style="color:#333333"><span style="background-color:#FFFFFF">Purify the DNA library by agarose gel electrophoresis</span></span></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s924" data-index="22" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s925"><ol class="steps"><li value="21"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s925" data-index="23"><span style="color:#333333"><span style="background-color:#FFFFFF">Recover the DNA library using a gel purification kit.</span></span></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s925" data-index="23" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s926"><ol class="steps"><li value="22"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s926" data-index="24"><span style="color:#333333"><span style="background-color:#FFFFFF">Quantify the DNA library by agarose gel electrophoresis</span></span></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s926" data-index="24" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-header" data-scroll-id="s927"><ul class="headers"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s927" data-index="25">NOTES</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s927" data-index="25" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ul></div><div class="mediocre-item is-step" data-scroll-id="s928"><ol class="steps"><li value="23"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s928" data-index="26"><span style="color:#8E2555"><span style="background-color:#FFFFFF">Primer should be complemetary and have similar melting techniques: </span></span><a href="http://bioweb.uwlax.edu/GenWeb/Molecular/seq_anal/primer_design/primer_design.htm" rel="nofollow" target="_blank"><span style="color:#8E2555"><span style="background-color:#FFFFFF">http://bioweb.uwlax.edu/GenWeb/Molecular/seq_anal/primer_design/primer_design.htm</span></span></a></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s928" data-index="26" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s929"><ol class="steps"><li value="24"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s929" data-index="27"><span style="color:#333333"><span style="background-color:#FFFFFF">Optimal PCR conditions should be determined prior to the use of this mutagenic PCR protocol. The doubling efficiency for the normal PCR reaction should be ∼1.7–1.9 per PCR cycle </span></span><span style="color:#333333"><span style="background-color:#FFFFFF"> and can be analyzed by running the PCR product after each cycle on an agarose gel</span></span></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s929" data-index="27" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s930"><ol class="steps"><li value="25"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s930" data-index="28"><i><span style="color:#333333"><span style="background-color:#FFFFFF">Taq</span></span></i><span style="color:#333333"><span style="background-color:#FFFFFF"> DNA polymerase should be stored at −20°C and kept on ice or in a frozen metal block when taken out of the freezer. The hot start procedure refers to adding the </span></span><i><span style="color:#333333"><span style="background-color:#FFFFFF">Taq</span></span></i><span style="color:#333333"><span style="background-color:#FFFFFF"> DNA polymerase to the PCR reaction mixture after the mixture has reached the annealing temperature. Either standard or “hot start” </span></span><i><span style="color:#333333"><span style="background-color:#FFFFFF">Taq</span></span></i><span style="color:#333333"><span style="background-color:#FFFFFF"> DNA polymerase will work in the described method.</span></span></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s930" data-index="28" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s931"><ol class="steps"><li value="26"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s931" data-index="29"><span style="color:#333333"><span style="background-color:#FFFFFF">It is important to prepare a fresh MnCl</span></span><sub><span style="color:#333333"><span style="background-color:#FFFFFF">2</span></span></sub><span style="color:#333333"><span style="background-color:#FFFFFF"> solution for the error-prone PCR experiment. This solution should be stored on ice and combined with the PCR reaction mixture at the start of each serial dilution.</span></span></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s931" data-index="29" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s932"><ol class="steps"><li value="27"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s932" data-index="30"><span style="color:#333333"><span style="background-color:#FFFFFF">It is important to prepare a fresh MnCl</span></span><sub><span style="color:#333333"><span style="background-color:#FFFFFF">2</span></span></sub><span style="color:#333333"><span style="background-color:#FFFFFF"> solution for the error-prone PCR experiment. This solution should be stored on ice and combined with the PCR reaction mixture at the start of each serial dilution.</span></span></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s932" data-index="30" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s933"><ol class="steps"><li value="28"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s933" data-index="31"><span style="color:#333333"><span style="background-color:#FFFFFF">The method described here was optimized for a 400-nucleotide gene. We highly recommend that experimenters read ref. </span></span><a href="https://link.springer.com/protocol/10.1007/978-1-60761-652-8_7#CR7_7" rel="nofollow" target="_blank"><u><span style="color:#333333"><span style="color:#8E2555"><span style="background-color:#FFFFFF">7</span></span></span></u></a><span style="color:#333333"><span style="background-color:#FFFFFF"> for information on the predicted average number of doublings and mutations expected as a function of template length</span></span></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s933" data-index="31" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s934"><ol class="steps"><li value="29"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s934" data-index="32"><span style="color:#333333"><span style="background-color:#FFFFFF">Agarose gel electrophoresis should be performed by standard methods. A DNA ladder with an appropriate base pair range should be used to identify and estimate the quantity of the PCR product.</span></span></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s934" data-index="32" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s935"><ol class="steps"><li value="30"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s935" data-index="33"><span style="color:#333333"><span style="background-color:#FFFFFF">It is recommended to save a portion of the PCR product from each tube for further analysis or amplification.</span></span></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s935" data-index="33" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s936"><ol class="steps"><li value="31"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s936" data-index="34"><span style="color:#333333"><span style="background-color:#FFFFFF">The concentration of the DNA product can be determined by comparing the intensity of the product bands to the intensity of the bands from the DNA quantification ladder from commercial sources.</span></span></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s936" data-index="34" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-header" data-scroll-id="s937"><ul class="headers"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s937" data-index="35">Transformation of Libraries </div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s937" data-index="35" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ul></div><div class="mediocre-item is-step" data-scroll-id="s938"><ol class="steps"><li value="32"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s938" data-index="36">GIbson Assembely can be performed if the primers of the linearized vector are complimentery to the error-prone PCR product.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s938" data-index="36" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-image" data-scroll-id="s939"><div class="image-editable"><form class="add-image-form"><input accept="image/*" class="image-input hidden" type="file"/><div class="upload-wrapper multi-editable"><img class="img" src="assets/proto-image-6deQnbrh4mWOszXlP3WfJEmOMdNtdLEdCtFAX5FS-Screen Shot 2018-05-17 at 2.19.41 PM.png"/></div><div class="progress-bar" style="width: 0%;"></div></form></div></div></div></div></div></div></div></div></div></div></div><!-- react-empty: 721 -->
+<div class="column half_size"style="width:70%;margin-left:15%">
+<h4>References</h4>
+<ol id="noIndent">
+<li>Random Mutagenesis by Error-Prone PCR
+McCullum E Williams B Zhang J Chaput J
+Methods in molecular biology (Clifton, N.J.)
+vol: 634 pp: 103-109</li>
+</div></div></div>
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Difference between revisions of "Team:Queens Canada/error2"

Latest revision as of 22:53, 13 October 2018

Error Prone PCR

Introduction

Materials

Procedure

References