Preparation:

Prepare SOB media and Transformation Buffer.

Sterilize 6, 250 mL centrifuge bottles; 100 mL graduated cylinder.

Pre-cool the rotor, buffer, and sterile plastic ware (above plus sterile 25 mL pipet, pipet tips, and 600 μL tubes) in the cold room.
 Set up 600 μL tubes in 1 mL pipet tip racks on ice during spins and incubations on the final day.
Day 1:
1.
Take 100 μL competent cells and inoculate a 10 mL SOB culture with selection.
Grow overnight at 37°C.
Day 2:
2.
Inoculate each of 6, 500 mL flasks containing 200 mL SOB (with selection) with
1 mL of the overnight culture. Grow at 18°C, shaking at 200-250 rpm until
OD
600
= 0.6. (This may take up to 3 days.)
Final Day:
First Spin -
3.
Transfer contents of each flask to a centrifuge bottle and balance them.
4.
Incubate bottles on ice for 10 minutes.
5.
Spin at 4100 rpm (2500 X G) and 2°C for 10 minutes in rotor coded 28, Sorvall
RC 5C Plus centrifuge.
6.
Place bottles on ice and take to cold room.
First Resuspension – IN COLD ROOM
7.
Pour off supernatant into flasks and invert bottles on paper towels.
8.
Gently resuspend pellets (by swirling) in 67 mL (1/3 original volume)
Transformation Buffer.
9.
Incubate on ice 10 minutes.
Second Spin
10.
Spin down cells exactly as before.
11.
Place bottles on ice and take to cold room.
Final Resuspension – IN COLD ROOM
12.
Pour off supernatant into flasks as before.
13.
Resuspend pellets in 17 mL (1/12 original volume) Transformation Buffer.
14.
Add 1.19 mL DMSO (595 μL twice, 7% final concentration), swirling gently.
15.
Incubate on ice 10 minutes.
16.
Dispense 210 μL cells into pre-cooled 600 μL tubes.
17.
Freeze with liquid N, and store immediately in -80°C.

SOB Medium
2% Bacto-tryptone
0.5% yeast extract
10 mM NaCl (0.6 g/L)
2.5 mM KCl (0.18 g/L)
10 mM MgCl2 (MgCl
2
-6H
2
O 2.03 g/L)
10 mM MgSO4 (MgSO
4
-7H
2
O 2.46 g/L or anhydrous 1.35 g/L) pH to 7.0 with NaOH, autoclave and store at 4°C
Transformation Buffer
10 mM Pipes or Hepes (2.5 g/L Hepes)
15 mM CaCl
2
250 mM KCl (18.6 g/L) pH to 6.7 with KOH, then add…
55 mM MnCl2 (10.9 g/L)
Filter sterilize through a 0.45 pm filter, store at 4°C.
NOTES:
1.
Don’t try to pre-cool the DMSO. It will crystallize.
2.
After 72 hours, a culture of XL-10 Gold was 0.65 (10/11/02 prep).
Reference: Inoue, H., Nojima, H., Okayarra, H. (1990) High efficiency transformation of Escherichia coli with plasmids. Gene 96:23-28
Protocol last edited: 10/11/02 J Tomscha