high percentage agarose

peter via methods%40net.bio.net (by peter.ianakiev from gmail.com)
Mon Aug 11 15:56:38 EST 2008


On Aug 11, 4:21 pm, d... from no.email.thankstospam.net (DK) wrote:
> In article <42c986dd-0c96-4fa2-bbef-d79a4d751... from o40g2000prn.googlegroups.com>, rashmi <rashmi21... from gmail.com> wrote:
>
> >Hi there,
> >I am a PhD student working on Pneumocystis biodiversity. I cam across
> >your message posted on google groups, regarding high percentage
> >agarose gels and TBE concentration. One of the objective of my thesis
> >is to do typing by counting number of 10 base pair repeats in my PCR
> >products. The number of repeats can vary from 2 to 6. The sizes are
> >125, 135, 145, 155 and 165. I tried to run a 4% agarose gel on a tray
> >about 25 cm. But my gel was not properly formed. i will be highly
> >delighted if you could suggest me something, like the % of agarose I
> >should use, run time, gel lengh, voltage etc.
>
> This is definitely a job for acrylamide gel. Google a bit and you
> shall find.
>
> DK

I have done in the pas discrimination of 3 bp 126/129 allele on 6%
agarose. The trick was to boil agarose for long time  and to add some
urea in it while is boiling. So if you need to make 100 ml 6% agarose
start with 200 ml H2O +10ml TBE + 6g Agarose, when is  boiling add 1-2
g Urea, keep boiling until volume is reduced to 100 ml and immediately
pour without cooling. Don't worry about bubbles, they will come out.
One more thing - run the gel as fast as you can (high voltage /cm) .
Good luck


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