Troubleshooting PCR: smears

Steven Goldberg goldberg at bms.com
Mon Mar 17 09:03:31 EST 1997


In article <E6zEt3.CoM at fsa.bris.ac.uk>, Harry Witchel
<Harry.Witchel at Bristol.Ac.Uk> wrote:

> Hello Immortals of Bio-cloning --
>    I am having trouble interpreting my PCR reactions.  I sometimes get 
> smears of different apparent weights:
> 
> 1) 1 kb to 50 bases
> 2) 1 kb to 6 kb
> 3) 6 kb to the well
> 4) smear goes from well to 50 bases
> 
> Although it seems obvious that the reaction is not stringent enough, 
> there are so many ways of increasing stringency (lower cycle number, 
> raise annealing temp, raise denaturing temp, add less Taq, add less 
> primers, change Mg2+) I was wondering if anyone has any specific ideas as 
> to what causes these smears.  I sometimes get smears all the way to the 
> top when I am using a plasmid as template, and I often get smears when 
> using a previous PCR reaction as the template (when using nested 
> primers).
>    Many thanks,
>    Harry

You might want to look at an article by Bell and DeMarini, Nuc. Acids.
Res. 19:5079 which says that too many cycles (>35) can convert PCR
products to higher molecular weight fragments of a random size.  Using
more primer and/or higher temperatures for annealing can help alleviate
this problem if your reaction is very weak.

Hope this is of some use

Steve



More information about the Methods mailing list