Agarose gel electrophoresis on PCR product

L. Sumoy Sumoy at mbcg.uchc.edu
Sat Jun 4 13:18:30 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(...) 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
> rather than nice discreet bands.
If you do not phenol extract the reaction before precipitation you may
be ending with protein being present in the concentrated DNA that
messes the solubility of your PCR product and prevents it from running
into the gel.
I agree that a fraction of the reaction should show up on a gel; there
is no need to run the whole reaction. We use TAE buffer with our
agarose gels. I do not that that difference should matter.
> The second problem is that a further round of reamplification on the
> GeneCleaned product has never produced decent results. I can't understand
> why this should be.
We have reamplified PCR product from polyacrylamide bands extracted by
the Maniatis freezing and crushing method (no need to phenol extract or
precipitate, just PCR from the eluted supernatant resulting from the
incubation of the band in elution buffer).
As suggested by other replies, reamplifying PCR or cloned DNA products
works best starting off from highly diluted template (in the order of
pg or less).
Good luck.
Lauro




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