Steve,
I can give you no good theory behind this, but the way we do
electorelution is different.
While we run analytical *agarose* gels with 1X TBE, we always run
preparative *agarose* gels in 1X TAE (tris-acetate-edta) and then do
the electroelution in 1 or 0.5X TAE. The band seems to run out of the
gel in a reasonable time (one-half hour or so at 100 V).
Their are two reasons we do this:
1) We usually concentrate the electroeluted DNA by butanol extraction
followed by ethanol ppt. Borate comes out of solution as you
concentrate it using butanol, acetate does not.
2) It seems that DNA fragments run through borate do not ligate well;
it is for ligations that we are generally electoreluting. I have no
good reason why borate would do this. But a number of folks I know
have observed the same.
So no good theory, but maybe if you electroelute in TAE, you will
satisfy your supervisor *and* get experiments done in a reasonable
amount of time. I have not run TAE *acrylamide* gels however....
my 2 electrons,
Brian
Steven Sanyal wrote:
>> Hello,
>> I have a quick question regarding the principle behind electroelution. I
> usually run my polyacrylamide gels using 1x TBE, from which I often cut out
> bands and electroelute the fragments.
>> I have found that when I electroelute using 1x TBE in the dialysis bag and
> in the electrphoresis apparatus (in which I place my dialysis bag), the
> fragment is out of the gel in 20 minutes at most, at 100 V.
>> My reasoning has been that having the buffer surrounding buffer the same
> concentration as the buffer used to make the gel was necessary in order to
> prevent a chemical gradient from forming in addition to the electric one.
>> My supervisor on the other hand has argued that this is rubbish. He told
> me to use 0.5 x TBE in the electroelution apparatus and just TE in the
> dialysis bag. Using these instructions though, it takes me over an hour to
> get the fragment out, and even then, it does not always look like it is
> out.
>> If someone could explain the theoretical principles behind this procedure,
> I would be much obliged. As far as I am concerned, I will stick with what
> works for me, but I would like to know why.
>> --
> Regards
>> Steve
>>ssanyal at netcom.ca>http://www.geocities.com/SoHo/Lofts/1785
