re-amp of PCR -- problems

bckraev at aeolus.ethz.ch bckraev at aeolus.ethz.ch
Wed Jan 11 05:28:48 EST 1995


Andrew, You do not need to re-purify your product, however, it may help if
you use the following trick: electrophorese your band into a low-gelling temp
agarose ( by cutting a trough ahead of the band and filling it with LGT agarose
letting it harden on ice for 10 min) and finally electrophoresing for 5-10 min
at approx. 10 V/cm), cut it out with a razor or a sharp scalpel. Melt the
gel slice for 10 min at 65 C and estimate the volume. Try to estimate the DNA
concentration in the melted slice ( e.g. by dividing the amount, say, 50 ng,
by volume in ul ). The amount anywhere from 1 to 10 ng can be directly added
to the preassembled PCR reaction, which has been pre-heated to 90 C. Usually
25 cycles are enough to re-amplify. Hope this helps.
Sasha



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