based on Mike Nonet's protocol, found here:

Follow it to the letter and work in the cold room as much as possible.

For DH5alpha, the 28 hours at 20C that Mike gives is a good estimate.

OP50 grew faster, about 23 hours at 20C.

We used a shaker set to about 150rpm.

We used 250ml Centrifuge bottles with purple lids, re-autoclaved before use.

SOB recipe (Super-Optimal Broth)

(modified for our lab's reagents and to make a 250ml batch)

5g Bacto tryptone

1.25g Bacto yeast extract

0.125g NaCl

2.5ml 250mM KCl (1.864g to 100ml)

to 250ml with MilliQ Water


Add 2.5ml sterile 1M MgSO4 just before use

DH5alpha made on 12/16/15

Collected at OD600 of 0.633

Aliquoted 200-1000ul per tube, which was too much.

Froze in liquid nitrogen immediately.

Tested transformation efficiency:

100ul cells + 4 ul of pUC19 at 50pg/ul

Plated 1/10 cells and got a lawn

Plated 1/100 cells and got >1600 colonies

TE= 0.84x10^9cfu/ug

Use less than 100ul cells per transformation