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.