inhibitet PCR reaction

chovek69 via methods%40net.bio.net (by ivanoov from gmail.com)
Thu Nov 1 10:17:16 EST 2007


On Oct 31, 5:48 pm, "Omar Hamarsheh" <ohamars... from gmail.com> wrote:
> Dear Colleges,
>
> I am working with ITS1 PCR using Leishmania DNA samples extracted from
> filter papers, the samples where originated from skin scraping and
> contain blood. I used inhibitor control for each sample . few samples
> were worked and gave positive. most of the others show no PCR bands,
> the inhibition control sho also no band which mean that there is an
> inhibition of the PCR reaction probably caused from something in
> blood.
> I would like to ask colleges who are working with clinical samples if
> they have an inhibitted PCR reaction what could be done in this
> regard.
>
> thanks alot in advance for any help or suggestion.
>
> Omar Hamarsheh
>
> Charite school of Medicine
> Dorotheenstr. 96
> 10117 Berlin
> Germany

Hi I have quite a bit experience with blood inhibited PCRs. The main
inhibitor is the hem from the hemoglobine but there are others. The
best remedy so far is the addition of nonacetylated Bovine Serum
Albumine (BSA) from Sigma, in 0.1 to 0.8 µg/µL final concentration.
Put in the PCR mix and sing. It depends also from the Taq but in
general all works the same. There are other polymerases that are not
inhibeted from the blood but they are expensive. (see for example this
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=87622

Hope this helps
Regards




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