Phenol extraction - necessary in this case? - (Dec/07/2005 )
Hi, here's a trivial question:
I have performed a restriction reaction using 2 enzymes and BSA, but loaded only half of it on the gel for a Southern. The other half I have diluted to 200 ul for use in PCR reactions.
But now I want to concentrate again the diluted half (from 200 to 20-40ul) to load on a new gel. Do I have to phenol extract the enzymes and BSA, or can just directly precipitate with EtOH? Would the proteins be a problem?
Thanks.
hi
for separation on gel i think it's ok to do only etoh. But not 10% sure.
but i think you won't loose enough dna by doing phenol chlo to risk a "standard etOH" only
Thanks. I think I'll go with EtOH only, because I've also noticed that my phenol is a little pinkish. Fingers crossed! 
Well I never extract my PCR products before loading them on a gel, do you? I would even avoid the EtOH precipitation and go to the speedvac, no loss, and ready to be loaded.
if your phenol hanged of colour, that's due to hydroquinolones and that's change the pH...
I load PCR products directly, like you, but these samples are restriction reactions with a good deal of denatured enzymes and albumin in them. I was afraid they might impede normal precitiptation of DNA, I still have to find that out...
Well I never extract my PCR products before loading them on a gel, do you? I would even avoid the EtOH precipitation and go to the speedvac, no loss, and ready to be loaded.
I load PCR products directly, like you, but these samples are restriction reactions with a good deal of denatured enzymes and albumin in them. I was afraid they might impede normal precitiptation of DNA, I still have to find that out...
But even then... When I am gel purifying my restriction digest, I directly load the product in the gel. Even if there was BSA in the buffer. Did you precipitate DNA on the other half? Then except if you diluted this half in a buffer full of proteins, you should be able to concentrate with speed vac...