Soaking in nucleic acids into a PAGE gel

Karl Fischer tyr-2 at bones.biochem.ualberta.ca
Wed Jul 11 17:22:43 EST 2001


Hello all,

Our lab is currently using activity gels to evaluate a virally-encoded
DNA polymerase. For those of you who are not familiar with this
technique it involves electrophoresis of proteins through a 8-10%
polyacrylamide gel into which a template for DNA synthesis is added as
part of the casting mixture (in our case the template is sonicated
herring sperm DNA). The protein sample is resolved under low SDS
concentration (0.02%), under low voltage at 10°C. Following
electrophoresis proteins in the gel are renatured in situ by extended
cold washes in Tris-HCl followed by incubation of the gel at 37°C in a
synthesis cocktail containing one labelled dNTP and three 'cold' dNTPs.
Following synthesis of DNA in situ the free hot nucleotide is removed by
extensive washing with cold 5%TCA-1%PPi. Newly sythesized hot DNA at the
position in the gel to which the enzyme was resolved appears as a band
on autoradiography.

What a colleague is interested in trying is leaving the polynucleotide
template out of the gel matrix for the electrophoresis step and to soak
in a template post-electrophoresis. My questions are:
1) Is such a soak-in approach possible? If so what concentrations of
template and duration of soak are required?
2) Is there a size limitation for the template which can be effectively
soaked into the gel?

Any information/suggestions would be greatly appreciated.

Kind regards,

Karl

fischer_karl at hotmail.com
karlster at excite.com




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