Revision as of 07:21, 9 October 2018

Protocols

Electrophoresis

Gel Preparation

Mix 1g of agarose powder with 100ml of 1x TAE buffer and heat for 1 minute or until all agarose is dissolved.

Wait until it has cooled (not set), and add 1ul of GelRed into the mixture.

Pour the solution into a cast with an appropriate comb.

Leave to set.

Running the Gel

Mix 1ul of 1kbp DNA ladder with 6ul of loading dye (bromophenol blue) and 4ul of 1 x TAE buffer (total 6ul) and load onto first well.

Mix 5ul of PCR products with 1ul of loading dye and load into wells.

Run gel at 90V products with 1ul of loading dye and load onto wells.

Run gel at 90V for 45 minutes approximately.

Photograph gels under UV light.

Manual Typsin Digest

For Coommassie stained gells, de-stain gels by washing briefly with 200µL NH4HCO3 (Solution A) to make sure gel pieces are at the correct pH.

Add 200µL 50% Acetonitrile / 50% 100mM NH4HCO3 (Solution B) into each well. Vortex to mix and incubate for 10 minutes. Remove liquid.

Repeat step 2. Gel pieces should be clear at this stage. If they are still blue, repeat as necessary until colour is gone.

Wash for 5 min with 50µL of 100% Acetonitrile (Solution C) to dehydrate gel pieces. Vortex during incubation.

Remove Acetonitrile, then let air-dry for 10 min. The gel pieces should be noticeably shrunken and probably white.

Reduction and Alkylation: Cover gel pieces with 50µL 10mM DTT in 50mM NH4HCO3 (Solution D). Let proteins reduced for 45-60 min in 37^oC incubator.

Remove DTT solution and add 50µL of 55mM iodoacetamide in 50mM NH4HCO3 (Solution E). Incubate for 45 min in dark place at room temperature.

Remove iodoacetamide, discard.

Wash gel pieces with 200µL of NH4HCO3 (Solution A) for 5 min with vortexing before adding 100µL of 100% Acetonitrile (Solution C).

Remove liquid after 5 min, discard.

Wash gel pieces with 50µL 100mM NH4HCO3 (Solution A) for 10 min, then twice with 200µL 50% Acetonitrile / 50% 100mM NH4HCO3 (Solution B) for 10min.

Dehydrate with 100 µL 100% Acetonitrile (Solution C) for 5 min as above.

Remove remaining liquid and let the gel dry.

Trypsin Digestion: Prepare trypsin mix to final concentration of 13ng/µL in 50mM Ammonium bicarbonate. Add 30µL (or more if required) of mixed trypsin solution to cover the gel pieces. Allow 30 min for gel rehydration at 4^oC (on ice). Digest overnight at 37^oC.

Peptide Extraction

Transfer the digest solution supernatant (if any) into clean 0.65ml Eppendorf tubes.

To the gel pieces, add 50 µL of 50% acetonitrile / 2% formic acid, incubate 30 min. Spin, remove supernatant and combine with initial digest solution supernatant. Please note that total volume may vary depending on the gel sizes.

Vortex the extracted digests, speed vac to reduce volume to 10 µL. If the remaining volume is less than 10µL, use 2% formic acid to bring the volume up to 10 µL.

Spin at 14,000 rpm for 30 min to remove any micro particulates.

Transfer the supernatant to a fresh 0.65ml eppendorf tube for storage at 4^oC fridge OR directly into PCR plate for Mass spec for analysis.

Trypsin Preparation

Trypsin is prepared by adding 200µL of Resuspension Buffer into one vial containing 20ug of Proteomics grade trypsin.

Add 1.2ml of 50mM NH4HCO3 into each trypsin vial (final concentration of Trypsin is 16ng/µL). Mix thoroughly.

This will provide enough Trypsin for 46 digestions.

Buffers and Reagents

50x TAE Buffer

Tris Base (MW=121.1)	242.4g
EDTA Disodium Salt	18.61g
Glacial Acetic acid	57.1mL

Dissolved Tris base and EDTA in 700mL distilled water, then added glacial acetic acid. Additional water was added to bring final volume up to 1L. Diluted to 1x for use.

50x M (100mL)

Na₂HPO₄	17.75g
KH₂PO₄	17g
NH₄Cl	13.4g
Na₂SO₄	3.55g

Made up to 100mL with distilled water. Autoclaved for 15 minutes to sterilise.

ZY Media (400mL)

Tryptone	4g
Yeast Extract	2g

Made up to 400mL with distilled water. Autoclaved for 15 minutes to sterilise.

50x 5052 (100mL)

Glycerol	25g
Glucose	2.5g
Alpha Lactose	10g

Dissolved glycerol in 40mL of distilled water. Dissolved glucose and alpha lactose in 40mL of distilled water. Combined two solutions, adjusted final volume to 100mL. Autoclaved for 15 minutes to sterilise.

TB Buffer (500mL)

KCI(MW=74.5513)	9.3g
CaCl₂.2H₂O (MW=147.0146)	1g
PIPES (MW=302.4)	1.5g
MnCl₂.4H₂O	5.44g

Dissolved KCL, PIPES and CaCl2 in 300mL of Milli-Q water. pH adjusted to 6.7 using KOH. Dissolved 5.44g of MnCl2 in 100mL of Milli-Q water. Gradually added MnCl2 to the other solution. Final volume was adjusted to 500mL, filter sterilised with 0.22µm filter, then stored at 4°C

HEPES, pH 7.5 (200 mL)

HEPES	47.66g
KOH	As required

Dissolved Tris in 75% of total volume of Milli-Q water. Buffered pH to correct levels with HCL, then adjusted final volume to 200mL.

Tris-HCl, pH 8.0 (200ml)

Tris	24.228g
HCl	As required

Dissolved Tris in 75% of total volume of Milli-Q water. Buffered pH to correct levels with HCL, then adjusted final volume to 200mL.

1M Imidazole, pH 8.0 (400ml)

Imidazole	27.232g
HCl	As required

Dissolve imidazole in 75% of total volume of Milli-Q water. Buffered pH to correct levels with HCL, then adjusted final volume to 400mL.

1M EDTA, pH 8.0 (200ml)

EDTA	29.22g
NaOH	As required

Dissolved EDTA in 75% of total volume of Milli-Q water while adding NaOH to assist. Buffered pH to correct levels with NaOH, then adjusted final volume to 200mL.

Dithiotheritol 1M (5mL)

DTT

0.7715g

Dissolved DTT in Milli-Q water.

TE Buffer (100ml)

TRIS (1M) pH 8.0	1mL
EDTA(0.5M) pH 8.0	0.2mL

Combined reagents, adjusted final volume to 100mL with Milli-Q water.

Cas9 Buffer (100ml)

HEPES(1M) pH 7.5	2mL
KCl(1M)	15mL

Combined reagents, adjusted final volume to 100mL with Milli-Q water.

CaCl₂ 1M (100mL)

CaCl₂	11.098g
KCl(1M)	15mL

Dissolved CaCl2 in 100mL of Milli-Q water.

Ammonium Bicarbonate 100mM (400mL)

Ammonium Bicarbonate

3.164g

Dissolved ammonium bicarbonate in 400mL of Milli-Q water.

Ammonium Bicarbonate 50mM (200mL)

Ammonium Bicarbonate

0.791g

Dissolved ammonium bicarbonate in 200mL of Milli-Q water.

50% 100mM Ammonium Bicarbonate/50% Acetonitrile (200ml)

Ammonium bicarbonate 100mM	100mL
Acetonitrile	100mL

Combined reagents in Schott bottle.

Coomassie Destain (1L)

Acetic Acid

100mL

Mixed with 900mL Milli-Q water

Coomassie Fixing Solution (400mL)

Acetic Acid	40mL
Ethanol	200mL

Combined reagents and adjusted final volume to 400mL with Milli-Q water.

Iodoacetamide (DTT)
Add 15 mL of 100 mM ammonium bicarbonate to 153 mg of iodoacetamide to give a solution of 55 mM iodoacetamide - 15 mL of each is enough for up to 96 samples.

Peptide Extraction Solution - 2% Formic Acid/ 50% Acetonitrile Solution
Add 2000µL of formic acid and 50 mL of acetonitrile to 75 mL of ddH2O. 15 mL is enough for up to 96 samples.

Competent Cells

A sterilized plastic loop was used to gather 10-12 large (2-3mm in diameter) colonies from the plate. Inoculated to 150mL of SOB medium in a 1L flask and grown overnight at 18-22°C, 200-250 rpm.

A600 readings were between 0.2-0.8 when harvested. Cells were in mid log phase with A600 ~ 0.5.

The flask was placed on ice for 10 minutes after being removed from incubator.

Culture was transferred to a 50mL centrifuge tube and spun at 2,500 x g for 10 minutes at 4°C.

Supernatant was discarded and the tube was placed on ice.

Cells were resuspended in 1mL of ice-cold TB buffer, ensuring no clumps of cells remained and kept cold.

Cold TB-buffer was added to bring up volume up to 1/5th of the original culture volume (~30mL) and mixed gently and inverted 3 times.

Tube was incubated on ice for 10 minutes and centrifuged at 2,500 x g for 7 minutes at 4°C and the supernatant was discarded.

Cells were resuspended in ~1/20th of the original culture volume of ice cold TB-buffer.

930µL of cell suspension was added to pre-chilled 1.5mL Eppendorf tubes.

70µL of DMSO was added to the 930µL cell suspension and mixed gently while placed on ice.

100µL of the competent cell/DMSO mixture was added into fresh microcentrifuge tubes, labelled and snapped frozen with liquid nitrogen.

Steps 10-12 was repeated to the remaining for the rest of the suspension from step 9.

Heat Shock Transformation

Obtain competent cells from -80^oC.

Defrost gently on ice. 100痞 is sufficient for 2 transformations.

Add 1-10痞 of plasmid DNA/ ligation mix to each tube. Incubate on ice for 30 min.

Heat shock in 42^oC water bath for 30 seconds, then back on ice for 2 min.

Add 200痞 of SOC media to each tube, and incubate in the 37^oC shaker for 1h.

For each tube of cells, spread 50痞 onto one LB plate with appropriate antibiotic, and 100痞 onto a second plate, using aseptic technique. Place your plate upside-down in the 37^oC incubator.

Media and Plates

LB Media (400mL)

Tryptone	4g
Yeast Extract	2g
NaCl	4g

Made up to 400mL with Milli-Q Water in a 500mL Schott bottle. Autoclaved for 15 minutes to sterilize.

LB Agar (400mL)

Tryptone	4g
Yeast Extract	2g
NaCl	4g
Bacto Agar	6g

Made up to 400mL with Milli-Q water in a 500mL in a Schott bottle. Autoclaved for 15 minutes to sterilize. 400µL of antibiotic was added once the bottle was cool enough to handle. Plates were poured using antiseptic technique.

SOB Medium (400mL)

Tryptone	8g
Yeast Extract	2g
5M NaCl	0.8mL
1M KCl	1mL
1M MgCl₂	4mL
1M MgSO₄	4mL

Made up to 400mL with distilled water. Autoclaved for 15 minutes to sterilize.

SOC Medium

Tryptone	8g
Yeast Extract	2g
5M NaCl	0.8mL
1M KCl	1mL
1M MgCl₂	4mL
1M MgSO₄	4mL
2M D-Glucose	4mL

Made up to 400mL with Milli-Q water. Autoclaved for 15 minutes to sterilize.

1% Agarose Gel

DNA Grade Agarose	1g
1x TAE Buffer	100mL
GelRed	4µL

Mixed components in 250mL flask and microwaved until completely dissolved. Cooled slightly before 4µL of GelRed added.

M9 Glycerol Agar

M9 Salts (5x)	100mL
1M MgSO₄	2mL
1M CaCl₂	50µL
20% Glycerol	10mL
Milli-Q Water	113mL

The next reagents were added to the mixture via filter sterilization:

1% Thiamine HCl	15mL
Cas Amino Acids	=10mL

Plates were then prepared using aseptic technique.

M9 Glucose Agar

Agar solution was prepared by autoclaving 7.5g of agar in 250mL of water. The following reagents were combined and autoclaved before being aseptically added to the hot agar:

M9 Salts (5x)	100mL
1M MgSO₄	2mL
1M CaCl₂	50µL
20% Glucose	10mL
Milli-Q Water	113mL

The next reagents were added to the mixture via filter sterilization:

1% Thiamine HCl	15mL
Cas Amino Acids	10mL

Plates were then prepared using aseptic technique.

Plasmid Prep

Centrifuge at 13,200 rpm for 10 min of 1.8mL of overnight cultures in 2mL Eppendorfs.

Discard supernatant and add 1.9 mL of culture and centrifuge again at 13,200 rpm for 10 min.

Re-suspend pelleted cells in P1 Buffer (250痞).

Add 250無 of P2 buffer and invert 4-6 times (turns homogenous blue).

Add 350無 of N3 and mix by inverting (turns colourless).

Centrifuge at 10min 13,000 rpm to obtain a pellet.

Transfer supernatant in QIA Prep Spin Column by pipetting.

Centrifuge 30-60 sec - discard flow through.

Wash QIA Prep Spin Column with 0.5mL of PB.

Centrifuge for 30-60 seconds - discard flow through.

Wash spin column by adding 0.75 mL PE buffer.

Centrifuge for 30-60 seconds - discard flow through and centrifuge for a further 1 min.

Place QIA Prep Column in clean Eppendorf.

Elute DNA by adding 30痞 of water, stand for 1 min, centrifuge for 1 min.

Plasmid concentration is measured using NanoDrop 2000 spectrophotometer.

Protein expression

IPTG Induction

Re-transform restriction-digest screened plasmids according to the heat shock transformation protocol.

Screen plates for transformant colonies and place in 5mL of LB broth with appropriate antibiotic concentration at 37^oC with shaking until OD600¬ is 2.0 – 3.0.

Take whole volume and place in 45mL of LB broth in a sterile conical flask containing appropriate antibiotic concentration. Incubate at 37^oC with shaking until OD600 is 0.5-0.7.

Transfer 1M IPTG in a 1:1000 dilution into flask. Incubate at 15^oC with shaking overnight.

Cell pellets were collected through centrifugation at 12,000 rpm for 5 mins.

Cell pellets were resuspended in re-suspension buffer (Containing: 10% glycerol w/v, 50mM Tricene NaOH pH 8.0, 2mM MgCl2, 1mM DTT) and whole lysate was collected through a French Press for functional assays of operon protein products in lysate.

Auto Induction

Start with glycerol stock or freshly transformed plate.

Inoculate 2ml starter culture with a single colony from plate, grow at 37^oC with vigorous shaking to OD600 ~ 1.

Transfer to a larger culture (50ml of ZYM-5052 medium in 250ml flask) and grow at room temperature (or 18^o C) overnight ~ 200rpm. For small scale, same starter flask can be grown at room temperature for ~24h. Amount of protein expressed peaks at OD600 ~ 8-10.

Spin down 5000 x g for 20 min at 4^oC, discard supernatant.

Resuspend pellet in 1/10th of the original volume.

To run on NuPage Bis-Tris gels, make sample mixture which contains: 20 µL resuspended lysate, 20 µL milli-Q water, 16µL NuPage LDS sample buffer, and 5µL 1M DTT.

Boil for 20 min, then spin down max speed for 20 min. Load 10-20µL of supernatant on gels.

Run gel at 150V for 1.5h, then fix with Coomassie Fixing solution for 10 min, stain with Coomassie Stain Solution for 5 min and destain overnight with Destain Solution.

SDS-PAGE

Re-suspend pelleted bacterial cells in 200µL of Milli-Q-water

Transfer 50 µL of suspension into new Eppendorf tubes and combine with 50µL of 2xTruSep sample buffer.

Shear the cells using a Hamilton syringe.

Centrifuge the preparation for 3 minutes at 13,000 rpm.

Load 20 µL of the supernatant into gel.

Conduct electrophoresis at a constant voltage (200V) for 1 hour.

Coommassie Stain for ~30 minutes.

PCR

General mastermix recipe:
4 µL of 5x phusion buffer

0.4 µL dNTPs

0.6 µL of DMSO

0.2 µL of polymerase

11.8 µL of water

To this mixture, add:
1 µL of forward primer

1 µL of reverse primer

1 µL of template

Total volume = 20 µL

PCR settings:

A general program for PCR is as follows:

Initial denaturation at 98^oC for 30 seconds

Followed by 30 repeats of:
Denaturation – 98^oC for 10 seconds

Annealing – 60^oC for 10 seconds

Extension – 72^oC for 2 minutes

Final extension – 72^oC for 10 minutes

@@ Line 235: / Line 235: @@
                  <font face="Arial" font size="5" color="#808080"><center>50x M (100mL)</center></font>
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                      </tr>
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+				</center>
                  <font face="Corbel" font style="line-height:1.5" font size="3" color="#404040">Made up to 100mL with distilled water. Autoclaved for 15 minutes to sterilise.</font>
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                  <font face="Arial" font size="5" color="#808080"><center>ZY Media (400mL)</center></font>
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+				<center>
                  <table>
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                  </table>
+				</center>
                  <font face="Corbel" font style="line-height:1.5" font size="3" color="#404040">Made up to 400mL with distilled water. Autoclaved for 15 minutes to sterilise.</font>
                  <br>
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                  <font face="Arial" font size="5" color="#808080"><center>50x 5052 (100mL)</center></font>
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+				<center>
                  <table>
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                      </tr>
                  </table>
+				</center>
                  <font face="Corbel" font style="line-height:1.5" font size="3" color="#404040">Dissolved glycerol in 40mL of distilled water. Dissolved glucose and alpha lactose in 40mL of distilled water. Combined two solutions, adjusted final volume to 100mL. Autoclaved for 15 minutes to sterilise.</font>
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                  <font face="Arial" font size="5" color="#808080"><center>TB Buffer (500mL)</center></font>
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                          <td>5.44g</td>
                      </tr></table>
+					</center>
                  <font face="Corbel" font style="line-height:1.5" font size="3" color="#404040">Dissolved KCL, PIPES and CaCl2 in 300mL of Milli-Q water. pH adjusted to 6.7 using KOH. Dissolved 5.44g of MnCl2 in 100mL of Milli-Q water. Gradually added MnCl2 to the other solution. Final volume was adjusted to 500mL, filter sterilised with 0.22µm filter, then stored at 4°C</font>
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                  <font face="Arial" font size="5" color="#808080"><center>HEPES, pH 7.5 (200 mL)</center></font>
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                  <table>
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                  </table>
+				</center>
                  <font face="Corbel" font style="line-height:1.5" font size="3" color="#404040">Dissolved Tris in 75% of total volume of Milli-Q water. Buffered pH to correct levels with HCL, then adjusted final volume to 200mL.</font>
                  <br>
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                  <font face="Arial" font size="5" color="#808080"><center>Tris-HCl, pH 8.0 (200ml)</center></font>
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+				<center>
                  <table>
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                  </table>
+				</center>
                  <font face="Corbel" font style="line-height:1.5" font size="3" color="#404040">Dissolved Tris in 75% of total volume of Milli-Q water. Buffered pH to correct levels with HCL, then adjusted final volume to 200mL.</font>
                  <br>
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                  <font face="Arial" font size="5" color="#808080"><center>1M Imidazole, pH 8.0 (400ml)</center></font>
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+				<center>
                  <table>
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                  </table>
+				</center>
                  <font face="Corbel" font style="line-height:1.5" font size="3" color="#404040">Dissolve imidazole in 75% of total volume of Milli-Q water. Buffered pH to correct levels with HCL, then adjusted final volume to 400mL.</font>
                  <br>
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                  <font face="Arial" font size="5" color="#808080"><center>1M EDTA, pH 8.0 (200ml)</center></font>
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                  <table>
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                  </table>
+				</center>
                  <font face="Corbel" font style="line-height:1.5" font size="3" color="#404040">Dissolved EDTA in 75% of total volume of Milli-Q water while adding NaOH to assist. Buffered pH to correct levels with NaOH, then adjusted final volume to 200mL.</font>
                  <br>
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                  <font face="Arial" font size="5" color="#808080"><center>Dithiotheritol 1M (5mL)</center></font>
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                  <table>
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                  </table>
+				</center>
                  <font face="Corbel" font style="line-height:1.5" font size="3" color="#404040">Dissolved DTT in Milli-Q water.</font>
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                  <font face="Arial" font size="5" color="#808080"><center>TE Buffer (100ml)</center></font>
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                  <table>
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                  </table>
+				</center>
                  <font face="Corbel" font style="line-height:1.5" font size="3" color="#404040">Combined reagents, adjusted final volume to 100mL with Milli-Q water.</font>
                  <br>
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                  <font face="Arial" font size="5" color="#808080"><center>Cas9 Buffer (100ml)</center></font>
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+				<center>
                  <table>
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                  </table>
+				</center>
                  <font face="Corbel" font style="line-height:1.5" font size="3" color="#404040">Combined reagents, adjusted final volume to 100mL with Milli-Q water.</font>
                  <br>
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                  <font face="Arial" font size="5" color="#808080"><center>CaCl<sub>2</sub> 1M (100mL)</center></font>
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                  </table>
+				</center>
                  <font face="Corbel" font style="line-height:1.5" font size="3" color="#404040">Dissolved CaCl2 in 100mL of Milli-Q water.</font>
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                  <font face="Arial" font size="5" color="#808080"><center>Ammonium Bicarbonate 100mM (400mL)</center></font>
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+				<center>
                  <table>
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                  </table>
+				</center>
                  <font face="Corbel" font style="line-height:1.5" font size="3" color="#404040">Dissolved ammonium bicarbonate in 400mL of Milli-Q water.</font>
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                  <font face="Arial" font size="5" color="#808080"><center>Ammonium Bicarbonate 50mM (200mL)</center></font>
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+				<center>
                  <table>
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                  </table>
+				</center>
                  <font face="Corbel" font style="line-height:1.5" font size="3" color="#404040">Dissolved ammonium bicarbonate in 200mL of Milli-Q water.</font>
                  <br>
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                  <font face="Arial" font size="5" color="#808080"><center>50% 100mM Ammonium Bicarbonate/50% Acetonitrile (200ml)</center></font>
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+				<center>
                  <table>
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                  </table>
+				</center>
                  <font face="Corbel" font style="line-height:1.5" font size="3" color="#404040">Combined reagents in Schott bottle.</font>
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                  <font face="Arial" font size="5" color="#808080"><center>Coomassie Destain (1L)</center></font>
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                  <table>
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                  </table>
+				</center>
                  <font face="Corbel" font style="line-height:1.5" font size="3" color="#404040">Mixed with 900mL Milli-Q water</font>
                  <br>
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                  <font face="Arial" font size="5" color="#808080"><center>Coomassie Fixing Solution (400mL)</center></font>
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+				<center>
                  <table>
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                  </table>
+				</center>
                  <font face="Corbel" font style="line-height:1.5" font size="3" color="#404040">Combined reagents and adjusted final volume to 400mL with Milli-Q water.</font>
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              <h1><font face="Arial"><center><span style="font-weight:normal; font-size: 23pt">Media and Plates</span></center></font></h1><hr>
              <font face="Arial" font size="5" color="#808080"><center>LB Media (400mL)</center>
-                 <table>
+                 <center>
+				<br>
+				<table>
                      <tr>
@@ Line 633: / Line 669: @@
                          <td>4g</td></tr>
                  </table>
+				</center>
                  <font face="Corbel" font style="line-height:1.5" font size="3" color="#404040">Made up to 400mL with Milli-Q Water in a 500mL Schott bottle. Autoclaved for 15 minutes to sterilize.</font>
                  <br>
@@ Line 639: / Line 676: @@
                  <h1><font face="Arial"><center><span style="font-weight:normal; font-size: 23pt">LB Agar (400mL)</span></center></font></h1>
                  <br>
+				<center>
                  <table>
@@ Line 656: / Line 694: @@
                          <td>6g</td>
                  </table>
+				</center>
                  <font face="Corbel" font style="line-height:1.5" font size="3" color="#404040">Made up to 400mL with Milli-Q water in a 500mL in a Schott bottle. Autoclaved for 15 minutes to sterilize. 400µL of antibiotic was added once the bottle was cool enough to handle. Plates were poured using antiseptic technique. </font>
                  <br>
@@ Line 664: / Line 702: @@
                  <br>
                  <table>
+				<center>
                      <tr>
                          <td>Tryptone</td>
@@ Line 686: / Line 724: @@
                          <td>4mL</td>
                  </table>
+				</center>
                  <font face="Corbel" font style="line-height:1.5" font size="3" color="#404040">Made up to 400mL with distilled water. Autoclaved for 15 minutes to sterilize.</font>
                  <br>
@@ Line 692: / Line 731: @@
                  <h1><font face="Arial"><center><span style="font-weight:normal; font-size: 23pt">SOC Medium</span></center></font></h1>
                  <br>
+				<center>
                  <table>
@@ Line 718: / Line 758: @@
                          <td>4mL</td>
                  </table>
+				</center>
                  <font face="Corbel" font style="line-height:1.5" font size="3" color="#404040">Made up to 400mL with Milli-Q water. Autoclaved for 15 minutes to sterilize.</font>
                  <br>
@@ Line 724: / Line 765: @@
                  <h1><font face="Arial"><center><span style="font-weight:normal; font-size: 23pt">1% Agarose Gel</span></center></font></h1>
                  <br>
+				<center>
                  <table>
@@ Line 738: / Line 780: @@
                          <td>4µL</td>
                  </table>
+				</center>
                  <font face="Corbel" font style="line-height:1.5" font size="3" color="#404040">Mixed components in 250mL flask and microwaved until completely dissolved. Cooled slightly before 4µL of GelRed added. </font>
                  <br>
@@ Line 744: / Line 787: @@
                  <h1><font face="Arial"><center><span style="font-weight:normal; font-size: 23pt">M9 Glycerol Agar</span></center></font></h1>
                  <br>
+				<center>
                  <table>
@@ Line 764: / Line 808: @@
                          <td>113mL</td>
                  </table>
+				</center>
                  <br>
                  <font face="Corbel" font style="line-height:1.5" font size="3" color="#404040">The next reagents were added to the mixture via filter sterilization:</font>
-                 <table>
+                 <center>
+				<table>
                      <tr>
@@ Line 777: / Line 823: @@
                      </tr>
                  </table>
+				</center>
                  <font face="Corbel" font style="line-height:1.5" font size="3" color="#404040">Plates were then prepared using aseptic technique.</font>
                  <br>
@@ Line 786: / Line 833: @@
                      The following reagents were combined and autoclaved before being aseptically added to the hot agar:
                  </font>
+				<center>
                  <table>
                      <tr>
@@ Line 805: / Line 853: @@
                          <td>113mL</td>
                  </table>
+				</center>
                  <br>
                  <font face="Corbel" font style="line-height:1.5" font size="3" color="#404040">The next reagents were added to the mixture via filter sterilization:</font>
-                 <table>
+                 <center>
+				<table>
                      <tr>
@@ Line 818: / Line 868: @@
                      </tr>
                  </table>
+				</center>
                  <font face="Corbel" font style="line-height:1.5" font size="3" color="#404040">Plates were then prepared using aseptic technique.</font>
                  <br>

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