PCR question

Brian Foley brianf at med.uvm.edu
Thu May 11 16:49:37 EST 1995


Ahmet Arman (aa374885 at OAK.CATS.OHIOU.EDU) wrote:
: Hi netters!
: I am doing PCR using 60 bp template and 15 mer primers. My PCR conditions:
:   94 oC 1 min
:   54 oC 1 min
:   72 oC 1 min

	How many cycles???  Try taking aliquots out after 10, 15 and 20 
cycles.  I have found that after I generate significant levels of a nice 
band, the PCR then tends to start generating concatomers of that band, 
and then a smear.
	Often times a PCR that shows a nice band after 10 cycles shows 
only a smear after 20 cycles.  Also, you can reduce the amount of 
template DNA, reduce the amount of TAQ polymerase, and increase the 
amount of primers to help eliminate overproduction of product.

: I never get single sharp band and am getting smear DNA. I checked all possible
: things like change annealing temperature, reduce template and time period of
: denaturation and extention which affect my PCR however; I ever get single 60 bp sharp band. Is
: there anybody which has better idea to get single sharp band.

: every suggestion will be appreciated.


: A. Arman
: aa374885 at oak.cats.ohiou.edu

--
********************************************************************
*  Brian Foley               *     If we knew what we were doing   *
*  Molecular Genetics Dept.  *     it wouldn't be called research  *
*  University of Vermont     *                                     *
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