Materials
- GYT (glycerol, yeast extract, tryptone)
- • 10%(v/v) glycerol
- • 0.125% (w/v) yeast extract
- • 0.25% (w/v) tryptone
- DI water
- 10% Glycerol
Special Equipment
- Centrifuge
- Ice water bath
- Liquid nitrogen
Method
Important: All steps in this protocol should be carried out aseptically
- Inoculate: Prepare flask containing 50 ml of LB medium. Pick up a single colony of cells from plate (using a sterile toothpick) and swirl around inside flask. Incubate the culture overnight at 37°C with vigorous aeration (250 pm in a rotary shaker).
- Dilute and incubate: Inoculate two aliquots of 475 ml of prewarmed LB medium in separate 2-liter flasks with 25 ml of the overnight bacterial culture. Incubate the flasks at 37°C with agitation (300 cycles/min in a rotary shaker). Measure the OD-600 every twenty minutes (this step will take around 1.5-2 hrs).
- Rapidly cool culture: Once the OD-600 of the culture reaches 0.6-1.0 （recommends 0.4), rapidly transfer the flasks to the pre-made ice-water bath for 15-30 minutes. Swirl the culture occasionally to ensure that cooling occurs evenly. In preparation for the next step, place the centrifuge bottles in the ice-water bath as well.
Note: After this point, do not let your cells warm up past 4°C
Note: When harvesting cells by decanting, be very careful to not disturb the pellet– this could result in a much lower yield. If necessary, aspirate instead of decant the supernatant. Get someone to show you how to aspirate. Also, if the pellet seems loose, sometimes it is helpful to re-spin the cells down.
- Centrifuge 1: Transfer the cultures to ice-cold centrifuge bottles. Harvest the cells by centrifugation at 1000g (2500 rpm) for 15 minutes at 4°C. Decant the supernantant and resuspend the cell pellet in 500 ml of ice-cold DI water. Note: I think this should be done for each of the two 500ml cultures, i.e this is a 1:1 resuspension rather than a concentration by a factor of 2 .
- Centrifuge 2 (water): Harvest the cells by centrifugation at 1000g for 20 minutes at 4°C. Decant the supernatant and resuspend the cell pellet in 250 ml ice-cold DI water.
- Centrifuge 3 (water): Harvest the cells by centrifugation at 1000g for 20 minutes at 4°C. Decant the supernatant and resuspend the cell pellet in 10 ml ice-cold 10% glycerol.
- To spin down your pellet in 10 ml, it might be helpful to use 15-ml Falcon tubes instead of the round-bottom tubes.
- Centrifuge 4 (glycerol): Harvest the cells by centrifugation at 1000g for 20 minutes at 4°C. Carefully decant the supernatant and use a Pastteur pipette attached to a vacuum line to remove any remaining drops of buffer.
- Resuspend in GYT: Resuspend in 1 ml ice cold GYT. This is best done by gently swirling rather pipetting or vortexing.
- Measure OD: Measure the OD-600 of a 1:100 dilution of the cell suspension. (In the cuvette, mix 0.99 mL water and 0.01 mL cell suspension).
Note: The desired concentration is [math]\displaystyle{ 2.5 \times 10^{11} }[/math] cells per mL, giving [math]\displaystyle{ 1\times 10^{10} }[/math] cells per 40 [math]\displaystyle{ \mu }[/math]L. This corresponds to an OD-600 (after 100x dilution) of roughly 3.75. It is difficult to reach this value, but it is still important to know the concentration of cells to calculate efficiencies.
- Dilute the cell suspension to a concentration of 2 x 10^10 to 3 x 10^10 cells/ml (1.0 OD600 = approx. 2.5 x 10^8 cells/ml) with ice-cold GYT medium.
- Test for arcing: Transfer 40 ul of the suspension to an ice-cold electroporation cuvette (0.1-0.2 cm gap, on middle shelf next to electroporator) and test whether arcing occurs when an electrical discharge is applied. Place the cuvette in the green holder attached to the machine. Go to option 4, Pre-set protocols; choose bacterial; choose the correct choice for your size cuvette, probably the first option for a .1 cm cuvette. If arcing occurs, wash the remainder of the cell suspension once more with ice-cold GYT medium to ensure that the conductivity of the bacterial suspension is sufficiently low (<5 mEq).
- Storage: Store cells at -80°C until they are required for use. For storage, dispense 40 ul aliquots of the cell suspension into sterile, ice-cold .5 ml microcentrifuge tubes, drop into a bath of liquid nitrogen and transfer to a -80°C freezer. To remove the tubes from the liquid nitrogen bath, bring out into the hall along with a storage box, and pour the tubes and liquid nitrogen into the box. Once all the tubes are out, close the box most of theh way and let the liquid run out into the hallway. Try not to do this in the very center of the walkway!
To use frozen cells: Remove an appropriate number of aliquots of cells from the -80°C freezer. Thaw the tubes on ice.