HELP!!! PCR smears!

Bengt Oxelman bengt.oxelman at systbot.gu.se
Wed Jul 20 13:02:14 EST 1994


I am sequncing rDNA from diverse green plants. For a lot of them, there
have been few problems. Now I have some templates where the desired
fragment seems to be impossible to amplify. All I get is a 'smear' of
various lengths. My old, succesfully amplified templates works still
though. My standard method for DNA extraction includes SDS/Mercaptoethanol,
phenol/chloroform extraction and salt/ethanol precipitation. I have also
used RNAse and CsCl sometimes, but it apparently didn't affect the outcome
of the PCR reactions. 

For the problematical templates I have tried:
- Excessive dilution of template (up to 5000x) - no effect
- Fewer cycles - less smear, but still no sharp band
- Shorter cycles - less smear, but still no sharp band
- less primer (from 50 to 10 per 50ul rxn) - more smear, but still no sharp
band
- less dNTPs - (from 0.2 mM to 0.1 mM) even more smear, but still no sharp
band
- less dNTPs and less primer - more smear including larger fragments, but
still no sharp band
- other bufferts - no effect
- other polymerases - variation in the amount of smear

Of course, it could be as simple as that the primers just don't complement
at the priming sites, but I hold that as unlikely. Could excessive RNA in
the template be the cause?

Any suggestions and/or comments to this would be greatly appreciated!
Thanks!
-- 
Bengt Oxelman
Dept. of Systematic Botany
Carl Skottsbergs Gata 22
S-413 19 Goeteborg
SWEDEN
bengt.oxelman at systbot.gu.se 



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