Removal of PCR inhibitors

jkcarson at netspace.net.au jkcarson at netspace.net.au
Thu Nov 25 03:18:23 EST 1999


On Wed, 24 Nov 1999 13:03:41 GMT, jkcarson at netspace.net.au wrote:
Thanks for all the useful and helpful suggestions. Some of the things
we have tried and didn't think to mention (too close to the problem!)
were: already using Triton-X in the lysis buffer; eluting with water;
eluting with 10mM Tris pH 8 (no EDTA) and 1mM Tris (no EDTA). Taking
out the EDTA (results from to-day) have certainly improved matters and
we can get amplification with 6ul of template now - a definite
improvement but we'd like better. We have a few other permutations to
try and we'll let you know the outcome. It would appear that excess
buffer plus EDTA in the elutant hasn't helped. The alpha-casein option
we will follow up.
Jeremy Carson

>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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