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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