Troubleshooting PCR: smears

Harry Witchel Harry.Witchel at Bristol.Ac.Uk
Thu Mar 13 07:31:51 EST 1997


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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