Removal of PCR inhibitors

Hans milana100 at hotmail.com
Wed Nov 24 11:48:06 EST 1999


On Wed, 24 Nov 1999 13:03:41 GMT, jkcarson at netspace.net.au wrote:

We achieved pretty good results with adding casein to the GuSCN
containing buffer. The casein we used was Sigma, it appears that you
cannot use just any kind of casein. The method was described in the
march 1999 issue of the J of Clin Microbiology.
Mail me if you need to know more details.

Hans

>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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