"

From 2013.igem.org

Preparation of Competent Cells

1. Pick a colony from plate using a toothpick and drop toothpick into 5mL of SOB media. Let this starter culture incubate overnight at 37 celsius in the shaker.

2. Fill two flasks with 100mL of SOB media.

3. Innoculate one flask with 500uL of starter culture, and the other flask with 50uL of starter culture. The reason for using two flasks is to have a safety net, so that if one of the flasks overshoots the optimal OD reading, there is still one more to fall back on.

4. Place flasks in the 18 celsius shaker.

5. Keep checking the OD reading (about every 4 hours).

6. Once the OD reading of one of the solutions in the flasks reaches around 0.7, it is ready to move on to the next step.

7. Take flask out and put it in ice, this is very important. From this point onwards, everything should be on ice as much as possible. Leave flask in ice for 10 minutes

7. Pour half of the contents in the flask into one 50mL falcon tube and the other half in another 50mL falcon tube.

8. Centrifuge at 4 celsius for 10 minutes at 3000 RCF. decel speed should be 3.

9. Pour out supernatant liquid in both falcon tubes. Gently pour it out because the pellets at the bottom are quite sensitive.

10. Resuspend one of the pellets using TB buffer. Cut the tip off of the 1mL pipette and resuspend the pellet with 1mL of TB buffer. Slowly wash the pellet. once the first pellet is fully resuspended, add the resuspended solution into the second falcon tube. now resuspend this pellet the same way as the first. Add 39 mL of TB buffer using the motor pipetter.

11. Centrifuge at 3000 rcf, 4c, 3 decel speed, 10 minutes

12. Remove supernatant liquid gentry and resuspend the pellet in 4mL of TB buffer. Check OD and add more TB buffer so that OD reaches 1.614.

13. 0.07 x total volume in Falcon tube gets you how much DMSO to use.

14. Get liquid nitrogen in styrofoam box.

15. Aliquot 100uL of competent cells per 1mL microtube. Mark the top of the tube and dip the bottom in liquid nitrogen. Then, simply drop the microtube in. Repeat process as many times as possible.

16. Use styrofoam box with holes at the bottom to isolate just the aliquots. put aliquots in freezer box and let liquid nitrogen stay near sink and evaporate overnight.