Hi,

I have chemical competent DH5alpha from Invitrogen (kept at -80). I usually make our own chem. competent bacteria stock from this. Every time I need to make a new batch of bacteria, I take a bit from the frozen stock with a sterile pipet tip and streak on a plain LB/agarose plate, incubate at 37 ON-20h. But this time, bacteria does not grow on the plate. I took a fresh plate as well, it did not help. The original stock is new and I keep them only few seconds on ice while I streak the plate.

Do you have a clue why they don`t grow??

Cheers

There are two main reasons I can think of off the top of my head:

1) your bacteria are dead, call invitrogen and get a new batch (tons of whys, don't worry about it and just start with new stock.)

2) you accidentally picked up a plate that has some antibiotic added to it, check your plate and try again.