re-amp of PCR -- problems

Paul N Hengen pnh at fcsparc6.ncifcrf.gov
Wed Jan 11 16:10:07 EST 1995


In article <3f0bt0$iso at mserv1.dl.ac.uk> bckraev at aeolus.ethz.ch writes:

: 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

I just did this cool trick to reamplify a PCR product of ~200 bp. Since I have
one of my primers biotinylated, I took 10 ul of Dynal M-280 streptavidin coated
paramagnetic beads and washed them in 500 ul of binding buffer. I then loaded
this into a small syringe fitted with an 18 G needle. To recover a single PCR
amplified band, I stuck the needle into the band within a 10% polyacrylamide
gel and pumped the buffer and beads in and out. Since I had stained with SYBR
Green I and did this on top of a 254 nm transilluminator, I could see the green
dye go up into the syringe.  Later, I recovered the beads with magnets, washed
twice with 200 ul of TE, and resuspended the beads in 10 ul of sterile H2O. I
used 1 ul of these beads in a fresh PCR mix to reamplify the fragment. After
PCR, I centrifuged out the beads and ran 10 ul of the DNA on a gel. It took all
of 60 seconds to get the band I wanted and it worked like a charm :-) Also, I
can reuse the template!  No toothpicking for me ;-)

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* Paul N. Hengen, Ph.D.                           /--------------------------/*
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