#1  Competent Bacterial Cell Preparation for Transformation using Plasmid DNA


Purpose is to make competent cells for general transformation use

1. Make SOB : 40g tryptone, 10g yeast extract, 1.16g NaCl, 4g MgCl2,
5g MgSO4, 763g Kcl, top to 2 L d2H2O

2. Make TB (transformation buffer): 800ml d2H2O, 3.02g Pipes, 2.21g
caCl2, 18.64g Kcl, pH 6.7 with KOH pellets, 10.89g MnCl2, 18g
Dextrose, up to 1L d2H2o > Sterile filter it > store at 4C

3. Inoculate 4x3ml of SOB with 1 colony each. Allow one extra tube
without inoculation to check for contamination

4. Grow 6-8hrs at 37C. Save the 2 tubes that grew the best. Grow 6ml
SOB o/n ~14-16h

5. Inoculate 2x500ml culture with 375ul of each of culture #1 and #2 =
750ul cells total inoculated (use the 2 starter culture tubes that
grew the best)

6. Incubate cultures at 37C for ~2-3h checking A600 periodically
This will take ~2-3h 37C shaker, ~180rpm. Check at 1h, then at 2h,
then as needed.

7. When OD is 0.06 but not greater for a 150ul aliquot (blank spec
with fresh SOB), remove and pour culture into 2x250ml centrifuge
bottles.

Tip:  USE BRAND NEW BOTTLES TO AVOID CONTAMINATION. DO NOT USE BOTTLES
THAT HAVE SEEN OTHER BACTERIA!! EVEN IF AUTOCLAVED!

Tip: Usually you'll only be able to use one of your 500ml flask,
because the other usually grows too much.  Weird.  The amounts below
are for one 500ml yield. If you get lucky and both grow close to 0.06,
double the volumes needed below.

8.  Centrifuge for 10min 2500xg at 4C. Decant supernatant, careful the
pellet will be "soft" !

9. Meanwhile, Set up ~100 autoclaved eppie tubes in a rack or
container at –20C or –80C

10. Ressuspend pellet in 40ml TB (20ml per 250ml tube), swirling
manually. Incubate on ice for 30min. Repeat centrifugation. Decant
supernatant.

11. Pool 4 pellets by serially ressuspending one pellet in 10ml TB
then transferring to a second pellet, etc. Keep bottles on ice at this
point.

12. Add 750ul DMSO to each of the 2 bottles = 1.5ml total (filtered or
sterile DMSO). Swirl bottle while adding

13. Aliquot ~125ul to eppie vials  ~100 tubes yield >> flash freeze in LiqN2

14. store tubes at –80C. Do not let comp cells thaw