Agarose gel electrophoresis on PCR product
bernard at elsie.nci.nih.gov
Wed Jun 1 22:27:44 EST 1994
In article <joiner-010694163702 at joiner.med.upenn.edu>, joiner at pobox.upenn.edu (Michael Joiner) writes:
> I am using a fairly standard PCR protocol to produce DNA which I am then
> checking by running the reaction product on a 0.5X TBE 2% agarose gel,
> identifying the band of interest, then cutting this out and extracting the
> DNA with the GeneClean kit. I have two problems.
> First, before running on the gel, I reduce the volume of the PCR reaction
> product from 40 microliters by adding NaOAC to 300mM, 2 volumes of EtOH,
> precipitating and spinning, then resuspending the "pellet" in 10
> microliters of water to which I add 2 microliters of "Manniatis type II"
> loading buffer. However, many times this has produced a gel which looks as
> if much of the DNA has remained in the well and with considerable smearing
[some text deleted for brevity]
Maybe I'm missing something fundamental, but why do you want to concentrate
it so much before electrophoresis? 40 - 50 ul is 1 - 2 wells on a "normal"
sized gel rig. Unless your DNA is so dilute that you have to run it in an
especially narrow well then don't bother with the dilution - add 4 ul of
x10 TBE and 4 ul of loading buffer and split it between two wells. If the
salt in the PCR buffer is nasty then maybe dilute it a little further.
Basically it sounds as if your gel is way overloaded.
Bernard Murray, Ph.D.
bernard at elsie.nci.nih.gov (National Cancer Institute, NIH, Bethesda MD, USA)
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