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real time PCR inhibition control


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3 replies to this topic

#1 yaeli

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Posted 12 September 2010 - 06:00 AM

Hello,

I use real time PCR for the quantification of invironmental samples.
These samples contain many inhibitors and I wish to add a control test that varify the presence of inhibitors left
after the DNA purification.
I heard there is an "alien" probe sequence that is being used which suppose not to accure in any creature genomic DNA
known in the NCBI database.
I undersatnd that poeple use this sequence instead of there own probe sequence between the same specific primers they designed.

If anybody know how to get to these sequences or have any other good test for the persence of inhibitor I would love to hear about it.

Thank you so much!

#2 ElHo

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Posted 13 September 2010 - 12:25 AM

You could try a dilution series of your samples. PCR efficieny should increase with higher dilution factors as you will also dilute potential inhibitors.

#3 Maddie

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Posted 15 September 2010 - 06:32 AM

Yes, you need an IPC (internal positive control). If you don't want to make it yourself, you can buy a kit from ABI that has everything in it.
One of my student works on soil samples and I know how problematic the inhibitors are.
ABI also developed a TaqMan assay especially for this type of environmental samples (the Taq had an extra purification step so it's not contaminated by bacterial DNA).

Edited by Maddie, 15 September 2010 - 06:33 AM.

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#4 tea-test

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Posted 16 September 2010 - 11:36 PM

hi, maybe this paper is useful, a sequence for an internal positive control is included.

http://www.ncbi.nlm....pubmed/16524557
tea-test: The artist formerly known as Ned Land




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