phenol-chlorofom extraction - (Nov/10/2005 )
is it possible to still perform a phenol chloroform extraction on a sample (restriction digest) that has loading buffer in it? are there any adjustments that can be made or do i have to redo teh digest?
why do you want to do that?
Why there is loading buffer in your restriction digest?
If you load all your restriction digested plasmid in a gel ( to run everything you can combine slots/wells with tape) and then you can easily and safely isolate the DNA from gel with a column.
Sorry if I didn´t maybe I understand your question.
so i was just going to be sure that my restriction digest worked. the reason i'm not using just a regular gel extract is b/c i'm digesting a bac & the total size is too big for the columns. therefore, one of my options to isolate my linearized sample is do the phenol-chloroform extract. usually i just add all the loading buffer into my sample b/c i am able to do a gel extract, however, i forgot that that wouldn't work & i had already added the buffer. i really don't want to go through the whole digest again & thats why i wondered if the phenol-chloroform extract would work with the loading buffer in it.
Oh I see,
But that I never I don't know. Of course you can risk it. If it works, good, if it doesn't I am afraid you have to start all over agian.
Let us know if it works.
I can't think of any major problem, there isn't EtBr right?? I would dilute the sample with loading buffer before extracting, this also helps to increase your recovery (add a co-precipitant if your overall concentration is less than ~10ng/ul)
I can't think of a problem either, but make sure that you're working with ~100uL (you can dilute to this as beccaf22 mentioned). Volumes less than this make it difficult to isolate the aqueous phase.
Also, since you're working with BAC, which is huge, be very gentle w/ your mixing steps. Be sure to gently invert to mix as opposed to vortexing or you'll probably shear BAC.
Best of luck,