HELP!!! PCR smear!
lcallico at utkvx.utk.edu
Sat Jul 30 14:39:09 EST 1994
In article <30k0ds$106 at mserv1.dl.ac.uk> bengt.oxelman at systbot.gu.se (Bengt Oxelman) writes:
>From: bengt.oxelman at systbot.gu.se (Bengt Oxelman)
>Subject: HELP!!! PCR smear!
>Date: 20 Jul 1994 21:11:40 +0100
>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!
>Department of Systematic Botany
>Carl Skottsbergs Gata 22
>S-413 19 Göteborg
>bengt.oxelman at systbot.gu.se
>Dept. of Systematic Botany, University of Goteborg
>Carl Skottsbergs Gata 22, S-413 19 Goteborg
>Phone: +46 31 7732669
>Fax: +46 31 7732677
>Internet e-mail: bengt.oxelman at systbot.gu.se
Try increasing the annealing temperature. That worked for one of the genes we
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