Protocols

Agarose Gel Electrophoresis

Materials:

agarose     0.25g
TAE         25mL
GelRed        2uL
6× Gel loading Dye  
DNA marker      4ul

Methods:

1. Heat the mixture until no particle can be seen.
2. Add GelRed when the temperature drops to around 55℃.
3. Pour the solution into the gel casting tray with appreciate comb.
4. Place the gel in the electrophoresis chamber and add enough TAE Buffer.
5. Add 6× Gel loading Dye into the samples and load them. 130V, 15min of electrophoresis.
6. Staining and visualization.

Notes:

The concentration of gel affects the resolution of DNA separation. For a standard agarose gel electrophoresis, a 0.8% gives good separation or resolution of large 5–10kb DNA fragments, while 2% gel gives good resolution for small 0.2–1kb fragments. 1% gels is often used for a standard electrophoresis.

A-T Ligation

Materials:

PCR product         x ng
pClone007 Blunt Simple Vector 1ul
10×Topo Mix         1ul
ddH2O           up to 10ul

Size of inserted fragment (bp)	100-1000	1000-2000	2000-5000
x (ng)	10-50	50-100	100-200

Methods:

Keep materials at room temperature (22-30℃) for 1-5 min.

Notes:

1. The materials should not be put on ice after ligation.
2. The PCR product should use taq DNA polymerase. Otherwise they should be added into the taq DNA polymerase system and incubate in 37℃ for 10min.

BioBrick Ligation

Materials:

Linear vector DNA       20-100ng
Insert DNA            5:1molar ratio over vector
10× T4 DNA Ligase Buffer    2uL
T4 DNA Ligase         1 Weiss U
ddH2O           up to 20uL
Total volume          20uL

Methods:

1. Incubate 10 min at 22℃.
2. Use up to 5uL of the mixture for transformation of 50uL of chemically competent cells or 1-2uL per 50uL of electrocompetent cells.

Notes:

1. The electrotransformation efficiency may be improved by:
   • Heat inactivation of T4 DNA ligase at 65℃ for 10 min or at 70℃ for 5 min.
   • Purification of DNA.
2. The overall number of transformants may be increased by extending the reaction time to 1 hour.
3. If more than 2 Weiss U of T4 DNA ligase is used in 20uL reaction mixture, it is necessary to purify DNA before electrotransformation.

Chemical Transformation

Materials:

DH5α component cell DNA plasmid LB media The selection plates

Methods:

1. Retrieve 1 tube of E. coli DH5α component cell on ice.
2. Add 10ng DNA plasmid into the tube.
3. Place the mixture on ice for 30 minutes.
4. Heat shock in 42℃ water bath for 45 seconds.
5. Place the mixture on ice for 2 minutes.
6. Pipette 900μl of LB media into the mixture.
7. Incubate at 37℃ and 220 rpm for 1 hour.
8. Centrifuge 5000 rpm for 2 minutes.
9. Aspirate the supernatant liquid and resuspend the bacteria in LB media.
10. Add them to the selection plate.

Colony PCR

Materials:

2x Taq Master Mix 10μl
Primer F          1μl
Primer R          1μl
Bacteria cultures   1μl
dd H2O up to      20μl

Methods:

1. Keep materials on ice, pipette reagents into a 100μl Eppendorf tubes.
2. Gently mix the reaction. Do not vortex the tube.
3. Use the centrifuge to collect all liquid to the bottom of the tube.
4. Put Eppendorf tubes into the thermocycler and set up the program.

Program:

94℃ 5min 94℃ 30s 56℃ 30s 35 cycles 72℃ 60s/kb 72℃ 7min 8℃ ∞

Gel purification with Tiangen Gel Extraction kit

Materials:

Buffer BL
Buffer PN
Buffer PW
Buffer EB
Spin columns CA2
2mL Collection Tubes

Methods:

1. Excise the DNA fragment from the agarose gel with a clean shaver blade;
2. Weigh the gel slice in a 1.5ml microcentrifuge tube. Add 1 volumes of Buffer PN to the same volume of gel (0.1g gel slice is treated as100μL buffer PN).
3. Incubate at 65°C for 10 min (or until the gel slice has completely dissolved). To help dissolve gel, mix by inverting the tube several times every 2–3 min during the incubation.
4. Add 500μl buffer BL to a column, centrifuge at 12000rpm for 1 min and discard flow through before use to maximally activate the column.
5. After the gel slice has dissolved completely, wait until the sample returns to room temperature and apply the sample to the column, let stand for 2min; Centrifuge at 1200rpm for 1 min and discard the flow-through.
6. Wash the column by adding 600μl buffer PW and centrifuging for 60s. Discard the flow-through.
7. Repeat the last step.
8. Centrifuge for 2 min to remove the residual wash buffer.
9. Place the column in a clean 1.5ml microcentrifuge tube and add 30-40μl ddH2O to the center of the column; Let stand for 2min and centrifuge for 2min; (If necessary, repeat this step).

Gibson Assembly

Materials:

Linear vector plasmid   100ng
Insert fragment     10:1 mole ratio (fragment to vector)
2xSoSoo Mix     5μl
ddH2O         up to 10μl

Methods:

1. Keep materials on ice, pipette reagents into a 100μl Eppendorf tubes.
2. Gently mix the reaction. Do not vortex the tube.
3. Use the centrifuge to collect all liquid to the bottom of the tube.
4. Put Eppendorf tubes into the thermocycler and set up the program.

Program:

50℃ 1h 8℃ ∞

Golden Gate Assembly

Materials:

ddH2O       up to 20ul
T4 ligase buffer    2ul
vector plasmid    10^10
insert fragment   10^10 x2
BsaI        1ul
T4 ligase       1ul

The relative molecular weights of DNA per base pair is 650Da. The length of the DNA (fragment or plasmid) is L bp. Then the DNA amount needed is: Fragment: m=L bp×650g/(mol∙bp)×〖10〗^10/(6.02×〖10〗^23 〖mol〗^(-1) )≈m_fra ng Plasmid: m=L bp×650g/(mol∙bp)×〖10〗^10/(6.02×〖10〗^23 〖mol〗^(-1) )≈m_plas ng

Methods:

1. Keep materials on ice, pipette reagents into a 100μl Eppendorf tubes.
2. Gently mix the reaction. Do not vortex the tube.
3. Use the centrifuge to collect all liquid to the bottom of the tube.
4. Put Eppendorf tubes into the thermocycler and set up the program.

Notes:

The Plasmid and/or the fragment should obtain Golden Gate restriction site.

PCR Program:

37℃ 15min 16℃ 10min 10 cycles 37℃ 10min 65℃ 10min 80℃ 10min 12℃ ∞

PCR Using Phanta Super-Fidelity DNA Polymerase

Materials:

ddH2O     up to 20ul
5×SF buffer   4ul
dNTPs Mix   0.4ul
Primer F      0.8ul
Primer R      0.8ul
Template DNA  100ng
DMSO (optional) 4ul
Polymerase    0.4ul

Methods:

1. Keep materials on ice, pipette reagents into a 100μl Eppendorf tubes.
2. Gently mix the reaction. Do not vortex the tube.
3. Use the centrifuge to collect all liquid to the bottom of the tube.
4. Put Eppendorf tubes into the thermocycler and set up the program.

PCR Program:

95℃ 3min 95℃ 10s 56℃ 20s 33 cycles 72℃ 2min 72℃ 10min 4℃ ∞

Plasmid extraction

Purification of plasmid using TIANGEN pure low copy plasmid kit

Materials:

Buffer BL buffer P1 Buffer P2 Buffer P3 Buffer PW Spin columns CP3 2mL Collection Tubes

Methods:

1. Column equilibration: Place a Spin Column CP3 in a clean collection tube, and add 500 μl Buffer BL to CP3. Centrifuge for 1 min at 12,000 rpm (~13,400 × g) in a table-top microcentrifuge. Discard the flow-through and set the Spin Column CP3 back into the collection tube.
2. Harvest 1-5 ml bacterial cells in a microcentrifuge tube by centrifugation at 12,000 rpm (~13,400× g) in a conventional, table-top microcentrifuge for 1 min at room temperature (15-25°C), then remove all traces of supernatant by inverting the open centrifuge tube until all medium has been drained.
3. Resuspend pelleted bacterial cells in 250 μl Buffer P1. Note: Ensure that RNase A has been added to Buffer P1. No cell clumps should be visible after resuspension of the pellet.
4. Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 6-8 times. Note: Mix gently by inverting the tube. Do not vortex, as this will result in shearing of genomic DNA. If necessary, continue inverting the tube until the solution becomes viscous and slightly clear. Do not allow the lysis reaction to proceed for more than 5 min. If the lysate is still not clear, please reduce bacterial pellet.
5. Add 350 μl Buffer P3 and mix immediately and thoroughly by inverting the tube 6-8 times. The solution should become cloudy. Note: To avoid localized precipitation, mix the solution thoroughly, immediately after addition of Buffer P3.
6. Centrifuge for 10 min at 12,000 rpm (~13,400× g) in a table-top microcentrifuge. A compact white pellet will form.
7. Carefully transfer the supernatant from step 6 to the Filtration Columns CS (place the CS in a collection tube) by decanting or pipetting. Centrifuge for 2 min at 12,000 rpm (~13,400× g). Note: Avoid pipetting out the precipitate.
8. Transfer the flow-through in the collection tube to the Spin Column CP3 by decanting or pipetting. Centrifuge for 1 min at 12,000 rpm (~13,400× g). Discard the flow-through and set the Spin Column CP3 back into the Collection Tube.
9. Recommended: Wash the Spin Column CP3 by adding 500 µl Buffer PD and centrifuging for 1 min at 12,000 rpm (~13,400× g). Discard the flow-through and set the CP3 back into the Collection Tube. Note: This step is necessary to remove trace nuclease activity when using end A+ strains such as the JM series, HB101 and its derivatives, or any wild-type strain, which have high levels of nuclease activity or high carbohydrate content.
10. Wash the Spin Column CP3 by adding 600 µl Buffer PW (ensure the ethanol (96%-100%) has been added to Buffer PW) and centrifuging for 1 min at 12,000 rpm (~13,400× g). Discard the flow-through and set the CP3 back into the Collection Tube.
11. Wash Spin Column CP3 by adding 600 µl Buffer PW and centrifuging for 1 min at 12,000 rpm (~13,400× g).
12. Discard the flow-through, and centrifuge for an additional 2 min at 12,000 rpm (~13,400× g) to remove residual wash buffer PW. Note: Residual wash buffer will not be completely removed unless the flow-through is discarded before this additional centrifugation. We suggest open CP3 lid and stay at room temperature for a while. Residual ethanol from Buffer PW may inhibit subsequent enzymatic reactions.
13. Place the Spin Column CP3 in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 30-50 μl water (pH 7.0-8.5) to the center of the Spin Column CP3, let stand for 2-5 min, and then centrifuge for 2 min at 12,000 rpm (~13,400×g)

Notes:

1. Add the provided Rnase A solution to buffer P1, mix and store at 2-8℃
2. Check Buffer BL, P2 and P3 before use for salt precipitation. If necessary, dissolve the buffer by warming at 37 ℃ for several minutes.
3. Avoid direct contact Buffer P2 and P3, immediately close the lid after use.
4. All centrifugation steps are carried out at 12000rpm(~13,400×g) in table-tap microcentrifuge at room temperature (15-25℃)
5. After treated with Buffer BL, use the Spin column soon, otherwise long-term placement may affect the purifying effect.
6. Add ethanol to Buffer PW before use, check bottle tag for the adding volume.

Preparation of LB Medium

Materials:

Yeast extract       5g
Tryptone          10g
NaCl            10g
H2O           up to 1L
Agar powder (for agar plate)  20g

Methods:

1. Add reagents into an Erlenmeyer.
2. Autoclave at 121 °C for 20 min.

Preparation of Minimal Medium

Minimal Medium:

Notes:

1. Solvent of component I II are water; Solvent of component III IV is 1 mol/L HCl.
2. Component III decomposes easily under light. Please put it into Brown grinding reagent bottle.
3. Components I III and V need to be sterilized at 121°C separately to prevent reaction between component.