I am using the PGEM T easy vector, and I am not even getting many negative colonies. I followed the protocol my instructor gave me, although it wasn't very detailed. I did some googling and realized I did a few things not quite right, but nothing that I feel should make me get so little results. 1) I defrosted the cells a little bit faster by warming them with my fingers, although I kept it on ice most of the time. 2) I centrifuged at 7600rpm 3 min, took off a bit of supernatent to increase concentration. When I resuspended the pellet before plating, I probably caused some shearing with the pipet tip I used to resuspend. (What is the best way to resuspend the pellet?)
Another thing I can think of that might have gone wrong: when I plated the cells, I used an glass loop using 100% isoprop. to innoculate. Perhaps I did not burn off all the isoprop., which killed the cells? (How long should I keep it over the flame?).
I trying to make sure I do it properly when I do this over again.
Edited by thc, 08 May 2009 - 05:21 PM.