PCR smearing problems

HARDIES at THORIN.UTHSCSA.EDU HARDIES at THORIN.UTHSCSA.EDU
Tue May 16 17:05:10 EST 1995


Jo Caine writes:

> I am screening a quick clone cDNA library using PCR. I'm looking for a product 
at 850 bp. What I am getting is a 400bp band that is surrounded by smearing.
I have tried varying Mg, DNA and primer levels, different DNA polymerases and
cycle conditions(Low annealing temperatures and high(48'C)/low(25'C) 
temperature programs). The Tm of the oligos are 51 and 57'C.

The true Tm's in 1.3 mM Mg are likely to be more like 60, and 66 C.  Try
higher annealing temps.  At these low temps. you may be hairpinning one of 
primers and taking it out of the reaction.

> Would I expect to see both the 850 and 400bp bands at 48'C or do I need to 
remove the nonspecific 400bp band, by going to a higher temperature, before I 
will get specific amplification?.

> My supervisor believes that if the 850bp is not present at 48'C then raising 
the temperature will not give the desired product.

It's possible that raising the temp. will solve the problem, for the reason
given above.  If it's really true that one of the primers has a Tm below
48, then lowering the temp. is the thing to try.  Try 45 or 42; 25 is too
low and will introduce other extraneous problems.  If your primers are really
this far apart, there may be no temp. where they work well together; you may
have to redesign them.  If you'd like to email me the primer sequences, I
will advise you further.  Most of this stuff is better determined by
examining the primer sequences than by trail and error experiments.

> I am also screening a lambda cDNA library by PCR. I am getting high molecular 
weight smearing, where I expect again a 850bp band. Conditions for PCR are as 
above, with oligo Tm's at 56 and 53'C. 
Anymore ideas?

Most commonly, this comes from using way too much template DNA.  Alternatively,
overcycling or anything that promotes incomplete extension can cause
accumulation of a network migrating high on the gel or even stuck in the
slot.

Hope this helps.
Steve Hardies, Assoc. Prof. of Biochem., Univ. of Texas HSC at San Antonio
Hardies at uthscsa.edu




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