Denaturing DNA - (Sep/25/2008 )
I have a small and rather simple question. I need to electroporate bacteria with a double stranded DNA and a couple of protocols suggest to denature DNA at first (without any further detail). What conditions would you use? I thought of applying 95 degrees for about 3-5 minutes, but how to treat it thereafter? For electroporation the bacteria are at 0 degrees in 10% Glycerol. If I would now put hot DNA solution to the bacteria (I have to use 1-10ul per 50ul of dissolved bacteria) it would likely ruin the electroporation, or what is idea of it? On the other hand, if I would cool the DNA down it would anneal back, wouldn't it?
You probably can cool down your template on ice immediately after denaturation.
Just electroporate it right away if it's a plasmid. no big deal!