Team:ZJU-China/Protocol

1. Preparation of heat shock competent cells.

*The most important thing to remember is to keep the cell as cold as possible at all the time.

Preparation：

Autoclave sterilization：Erlenmeyer flask；1L reagent bottles (for TFB)；100ml centrifuge bottles (bottles and caps need to be sterilized separately)；vacuum filter flask; 0.22nm filter membrane；80%glycerol; LB broth.

Procedure:

1. Streak out E. coli (DH5α, DH10β, BL21, BW25113 and so on) onto LB plate. Cultivate it inversely.
2. Growing overnight @37℃
3. Pick a single colony and inoculate overnight using 5mL LB Broth, shake it @37℃, 200r/min.
4. Inoculate 2ml overnight culture to 100ml LB broth, shake it @37℃, 200r/min until OD600=0.4 or so. (About 3.5 hours)

   * Remember to open spectrophotometer in advance and use LB as blank.

5. Chill the cell on ice for 15min.
6. Centrifuge the cells at 5000rpm for 10min @4℃.

   *Remember to balance the centrifuge bottles/tubes before centrifugation.

7. Discard the supernatant.
8. Resuspend the cells with 2ml TFB. Then fill it with TFB.
9. Centrifuge at 5000rpm for 10min @4℃.
10. Add some TFB buffer to resuspend the cell, and then fill the tube with TFB buffer.
11. Centrifuge at 5000rpm for 10min @4℃.
12. Discard the supernatant.
13. Resuspend the cell with 3ml TFB buffer, then add 700ul 80%glycerol to 15% of final concentration of glycerol.
14. Fill the 1.5ml EP tube with 50ul liquid. Quick-freeze in liquid nitrogen. Stock in -80℃.

Preparation of TFB:

Mother solution: CaCl2: 0.5M, MgCl2: 1M.

Reagent	Final concentration	500ml
CaCl2	100mM	100ml
MgCl2	70mM	35ml
NaAc(add powder)	40mM	1.64g

Using acetic acid to adjust pH to 5.5, Filtrated, Stock @4℃.

2. Heat shock transformation.

Preparation:

Water bath 42℃, rotary shaker @37℃, 150rpm, ice, E. coli @-80℃.

Procedure:

1. Thaw the competent cells or super competent cells on ice. About 10 minutes.
2. Add 1ul of plasmids (about 30ng) to cells and swirl it gently.
3. Incubate the cells on ice for 30 minutes.
4. Heat pulse the tube in a 42℃ water bath for 80~90 sec.

   *The length of time of the heat pulse is critical for obtaining the highest efficiencies.

5. Incubate the cells on ice for 2min.
6. Add 1ml of LB medium to the tube. Then shake it @37℃ 150rpm for about 1h.
7. Centrifuge the tube at <5000rpm for 2min, then discard the supernatant making the residue less than 50ul.
8. Coat the plates containing specific antibiotics with the culture.
9. Cultivate the plate inversely @37℃ for about 12h (12h~14h).

3. Preparation of Electrocompetent Cells.

Preparation:

Autoclave sterilization：Erlenmeyer flask；1L ddH2O；1L 10% glycerol; 100ml centrifuge bottles (bottles and caps need to be sterilized separately)；LB broth.

Procedure:

1. Inoculate 5ml L-broth with a single colony of E. coli. Incubate 5 hours to overnight at 37°C on a roller or with moderate shaking.
2. Inoculate a volume of L-broth contained in an appropriately sized side-arm flask with one-tenth volume of the culture (i.e. 1 ml of culture to 100 ml L-broth). Grow cells at 37°C with shaking (200-300 rpm) to an OD600 of 0.55 to 0.6.
3. Chill the cells in an ice-water bath for 10 to 15 minutes and transfer to a pre-chilled centrifuge bottle. (Divide the culture if required.)

   *Cells should be kept at 2°C for all subsequent steps.

4. Pellet the cells by centrifugation at 5000 rpm, 4°C for 10 minutes.
5. Pour off the supernatant and resuspend the cells in 1mL ice-cold ddH2O. Add 100mL ice-cold ddH2O. Centrifuge the cells as in step 4.
6. Pour off the supernatant immediately and resuspend the cells in the small amount of fluid remaining in the bottle.

   *The pellet may be very loose. Exercise care and pour off the supernatant immediately.

7. Add 50mL of ice-cold WB (10% glycerol). Centrifuge the cells, again as in step 4. Pour off the supernatant immediately and resuspend the cells in the remaining fluid.
8. Place the cell suspension in an appropriately-sized, narrow-bottom tube that has been pre-chilled.
9. Add to the cells an amount of ice-cold 10% glycerol equal to 0.01 of original culture volume (1 ml for a culture of originally 100 ml) and mix well.
10. Aliquot 40 ul of the cells to pre-chilled EP tubes. Freeze the cells by incubation in a liquid nitrogen bath. Store at -80°C.

4. Electroporation of the Cells. (for plasmid)

Preparation:

0.1cm electroporation cuvettes; rotary shaker; ice; E. coli @-80℃.

Procedure:

1. Set the MicroPulser to Eco 1 state (2.5 kV, 25 µF). Add 1 µl plasmid DNA to tubes containing 40 µl fresh or thawed cells on ice. Mix by swirling with pipette tip.

   *The volume of DNA added to the cells should be kept small.

   *Adding DNA up to one-tenth the volume of cells can reduce the efficiency of electroporation 2- to 3-fold.

2. Transfer the DNA and cells to a pre-chilled electroporation cuvette (0.1 cm electrode gap) using a narrow pipette tip.
3. Wipe any ice or water from sides of cuvette using a Kimwipe. Place the cuvette into the sample chamber.
4. Energize MicroPulser and deliver the pulse by pushing in both charging buttons simultaneously and holding until a short beep is heard.

   *Note the time constant of the pulse and the actual voltage delivered.

5. Remove the cuvette from the sample chamber. Immediately add 1 ml SOC medium and transfer the cells to a sterile EP tube using a Pasture pipette.

   *Failure to immediately add SOC to the electroporated cells can significantly reduce cell viability and decrease transformation efficiency.

6. Incubate cultures for 1 hour at 37°C (if cells have pKD46, incubate them at 30°C) on a roller or with moderate shaking to allow for plasmid expression.
7. Plate 100uL of the electroporation mixture on L-agar plates supplemented with the appropriate antibiotics. Incubate plates at 37°C (if cells have pKD46, incubate them at 30°C).

Preparation of SOC Broth (1 liter)

Bacto tryptone	20.0 g
Bacto yeast extract	5.0 g
NaCl	0.6 g
KCl	0.5 g
MgCl2	10 mM
MgSO4	10 mM
Glucose	20 mM

Note: SOC is identical to SOB, except that it contains 20 mM glucose.

1. Dissolve tryptone, yeast extract, sodium chloride, and potassium chloride in a final volume of 970 ml distilled H2O. Sterilize by autoclaving.
2. After autoclaving, allow the solution to cool to 60°C, and add 20 ml of a sterile 1 M glucose stock (see below) to make the media 20 mM with respect to glucose.
3. Just prior to using, add 10 ml of magnesium stock (see below) to the SOC broth to make the media 20 mM with respect to magnesium.

5. Preparation of Electrocompetent and recombination-proficient Cells.

Preparation:

Autoclave sterilization：Erlenmeyer flask；1L ddH2O；1L 10% glycerol; 100ml centrifuge bottles (bottles and caps need to be sterilized separately)；LB broth; 50mL Arabinose.

Procedure:

1. Inoculate 5ml L-broth (Amp) with a single colony of E. coli with pKD46. Incubate overnight at 30°C on a roller or with moderate shaking.
2. Inoculate a volume of L-broth (Amp) contained in an appropriately sized side-arm flask with one-tenth volume of the culture (i.e. 1 ml of culture to 100 ml L-broth). Grow cells at 30°C with shaking (200-300 rpm) to an OD600 of 0.2 or so.
3. Add L-arabinose to the culture (Final concentration is 30mM. We’ve done a gradient analysis of arabinose concentration of this step.)
4. Chill the cells in an ice-water bath for 10 to 15 minutes and transfer to a pre-chilled centrifuge bottle. (Divide the culture if required.)

   *Cells should be kept at 2°C for all subsequent steps.

5. Pellet the cells by centrifugation at 5000 rpm, 4°C for 10 minutes.
6. Pour off the supernatant and resuspend the cells in 1mL ice-cold ddH2O. Add 100mL ice-cold ddH2O. Centrifuge the cells as in step 5.
7. Pour off the supernatant immediately and resuspend the cells in the small amount of fluid remaining in the bottle.

   *The pellet may be very loose. Exercise care and pour off the supernatant immediately.

8. Add 50mL of ice-cold WB (10% glycerol). Centrifuge the cells, again as in step 4. Pour off the supernatant immediately and resuspend the cells in the remaining fluid. Repeat this step one more time.

   *The supernatant should be poured off completely. We can use pipettes to achieve it.

9. Add to the cells 100uL ice-cold 10% glycerol mix well.

   *The competent cells need to be concentrated enough.

10. Aliquot 40 uL of the cells to pre-chilled EP tubes. Freeze the cells by incubation in a liquid nitrogen bath. Store at -80°C. Or use the cells freshly.

6. Electroporation of the Cells (for dsDNA)

Preparation:

0.1cm electroporation cuvettes; rotary shaker; ice; E. coli @-80℃.

Procedure:

1. Set the MicroPulser to Eco 1 state (2.5 kV, 25 µF). Add 5µl dsDNA (about 1000ng) to tubes containing 40 µl fresh or thawed cells on ice. Mix by swirling with pipette tip.

   *The volume of DNA added to the cells should be kept small.

   *The mass of dsDNA added should be large enough. (about 1000ng)

2. Transfer the DNA and cells to a pre-chilled electroporation cuvette (0.1 cm electrode gap) using a narrow pipette tip.
3. Wipe any ice or water from sides of cuvette using a Kimwipe. Place the cuvette into the sample chamber.
4. Energize MicroPulser and deliver the pulse by pushing in both charging buttons simultaneously and holding until a short beep is heard.

   *Note the time constant of the pulse and the actual voltage delivered.

5. Remove the cuvette from the sample chamber. Immediately add 1 ml SOC medium and transfer the cells to a sterile EP tube using a Pasture pipette.

   *Failure to immediately add SOC to the electroporated cells can significantly reduce cell viability and decrease transformation efficiency.

6. Incubate cultures for 1 hour at 37°C (The cells may lose pKD46.) on a roller or with moderate shaking to allow for plasmid expression.
7. Centrifuge the cells to get liquid volume less than 100uL.
8. Plate all of the electroporation mixture on L-agar plates supplemented with the 1/2 Ampicillin. Incubate plates at 37°C.
.

7. Overlap PCR

Preparation:

Two fragments with 10~20bp overlap each other.

Procedure:

1. To amplify the final fusion product,use the outside primers and a 1:1 mix of the 3’ and 5’ fragments amplified in the initial PCR reaction.

   Adjust each PCR product to about 80ng/ul with water(for genome).Assemble a PCR reaction as follows:

2. example: 10 ul 5X buffer

   4 ul dNTPs (4 mM)

   1 ul front Primer (10 uM)

   1 ul reverse Primer (10 uM)

   1 ul 1:1 mix of 5’& 3’fragments at ~80ng/ul each(for genome)

   0.25 ul FASTpfu polymerase

   1 ul genome DNA

   Add H20 to 50ul

3. Example: 1 hold: 94 oC,5 min

   30 cycles: 94 ℃,30 sec

   X ℃,30 sec (adjust according to specific primer Tm’s)

   72 ℃, X min (adjust according to predicted fusion size)

   1hold: 72℃,5min

   1hold: 4℃

4. Visualize the PCR reaction by loading 5 ul on a gel.

   If the target band is dominant, purify the PCR product using the clean-up kit.

8. Instructions

1. AxyPrep DNA Gel Extraction Kit
2. AxyPrep Plasmid Miniprep Kit
3. Thermo Scientific GeneJET RNA Purification Kit
4. Thermo Scientific RevertAid RT Kit
5. EcoRI
6. HindIII
7. PstI
8. XbaI
9. PrimerSTAR HS DNA Polymeras
10. T4_DNA_Ligase
11. Taq Full DNA Polymerase User Manual
12. TaKaRa LA Taq DNA Polymerase
13. GBclonart Seamless Assembly Kit
14. GBclonart Seamless Cloning Kit

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