Troubleshooting PCR: smears

Baylor Molecular Genetics Lab Steven_Burghart at baylor.edu
Thu Mar 13 11:14:51 EST 1997


Hi Harry
I am not expert but hope this will help.
Sometimes original DNA degradation could cause these smears.
Also it is dependent from your subject (species). I used PCR for taxonomy
of different plant species and quality of these species were different,
from extremely good to extremely bad. If you need to clone PCR product
treat first by GeneClean Kit. Ideally cloning/transformation should be
done within one-two days.
Hope this will help. Good luck. Yerlan  

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

-- 
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