re-amp of PCR -- problems

[Michael Cooley]

zool201 at csc.canterbury.ac.nz wrote:
: To whom it may concern

: I'm trying to reamplify a very low concentrartion of primary PCR
: product and am ending up with smears every time.
: I don't want ot re-purify the primary product because of the very low 
: concentration.

: Any hints would be greatfully accepted.

: Andrew Holyoake 
: PhD student
: University of Canterbury
: Christchurch 
: New Zealand

: I am trying to reamplify very low concentration primary PCR






This problem is not uncommon. It is probably caused by non-specific 
amplification in the original amplification. Gel purification will help, 
but some times it doesnot work. The best cure is to redo the orginal 
amplification at higher strigency even if it decreases the yield of the 
specific product. The low melting agarose trick discribed above will 
work, but an easier approach is to stick a sterile toothpick into the 
gel at the site of the fragment, then stir it into the new PCR reaction 
mix. Also I suggest, at least, increasing the strigency of this second 
reaction. Cetus suggests also other cures; decrease the number of cycles, 
decrease the amount of enzyme, decrease the amount to dNTPs, but the 
obvious solution is to increase the annealling temp or decrease the Mg level.


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