Removal of PCR inhibitors

jkcarson at netspace.net.au jkcarson at netspace.net.au
Wed Nov 24 08:03:41 EST 1999


We have been using the guanidinium thiocyanate - diatom method for
extracting bacterial DNA and has been working well. The extracted DNA
is used for a 16S rRNA amplification. Our PCR reaction volume is 20ul
and we use a 1ul template volume. We have modified the PCR format so
that we can use 9ul of template in the 20ul PCR format - a strategy to
increase sensitivity. We validated the modification using DNA purified
by phenol-chloroform.
If we use a 9ul volume of  template DNA prepared using the guanidinium
method we do not get any PCR product but if we use a 1ul volume as
template the PCR works. We have concluded that an inhibitor is
co-extracting. BSA does not neutralise the effect. 
Does anyone have any suggestions on how to neutralise the PCR
inhibitor? Has anyone tried GeneReleaser for post-extraction cleanup
or used alpha-casein to neutralise PCR inhibitors. For various reasons
we are locked in to the guanidinium-diatom method of DNA extraction.
Thanks
Jeremy Carson
Dept Primary Industry
Tasmania, Australia
jeremy.carson at dpiwe.tas.gov.au




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