Discountinuous denaturing PAGE of DNA oligonucleotides

Giulio Casi gcasi at inwind.it
Mon Apr 5 10:13:10 EST 2004


Hello everybody,

I have to run PAGE of few oligonucleotides under denaturing conditions, and
I am facing few problems:

1- The bands look quite fat
2- The run time is quite high (>3h)

The exact conditions I am using are:

1- DNA 12-mer
2- 300 V
3- 24 % Polyacrylamide homogeneous gel; 8M urea; 1x TBE
4- 15 cm gel lenght
5- Loading Buffer: 70% Formamide 30% Bromophenolblue

I tried to make a discontinuous gel (4% polyacrylamide and then 24%) but it
resulted in
thikker bands, compared to homogeneous gels.

Did anybody of you ever run a discontinuous gel for oligonucleotides? Does
anyone know a possible solution in order to have thinner bands, and shorter
run times?
I thank you all in advance for your help

Sincerely,
Giulio Casi

gcasi at inwind.it





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