PCR smearing problems

JUDITH-C at staff.monash.edu.au JUDITH-C at staff.monash.edu.au
Tue May 30 04:51:58 EST 1995


In article <950516170850.20207338 at thorin.uthscsa.edu> HARDIES at THORIN.UTHSCSA.EDU writes:
>Path:
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>From: HARDIES at THORIN.UTHSCSA.EDU
>Newsgroups: bionet.molbio.methds-reagnts
>Subject: re:PCR smearing problems
>Date: 16 May 1995 15:05:10 -0700
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>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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