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PCR on "dirty" samples

PCR on "dirty" samples - PCR - Polymerase Chain Reaction Forum

PCR on "dirty" samples - PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory.


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  #1  
Old 03-31-2008, 08:12 PM
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Default PCR on "dirty" samples



Hi all!

I'm getting very tiny biopsy samples for my PCR analysis. I tried to purify DNA from these samples using conventional methods, adding glycogen, etc, but never got any detectable DNA to work with. I ended up incubating my samples in lysis buffer @95C for 10min, spinning them down, and using supernatant to run PCR. Sometimes this method works and I get PCR products, sometimes it does not. My guess is that I have contaminant(s) that inhibits PCR.
Does anybody have any good suggestions on how to clean up my samples?
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Old 03-31-2008, 10:21 PM
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Default Re: PCR on "dirty" samples

Hello Elena,
sounds like partly dirty samples and partly low yield from the purification.

I would try pcr from pcr. You could do this by first pcring your samples as you said, and then taking 1ul of pcr and using that in your pcr.

Also try diluting it 1/10 and taking 1ul for pcr in case you do have a contaminant.

Cleaning up your samples may also be the problem as your DNA may be very little. Try concentrating it and purifying it with a qiagen column for pcr purification. Then try using that for pcr.

tell us how it went.

cheers
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Old 04-01-2008, 07:28 PM
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Default Re: PCR on "dirty" samples

Try a DNA extraction protocol or a kit. Just boiling and using supernatant, is not a good strategy, your lysis buffer itself (especially any EDTA or high salts) may contain the PCR/Taq inhibitor.
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Old 04-01-2008, 07:40 PM
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Default Re: PCR on "dirty" samples

Also Clontech Chromaspin-100 columns are excellent at removing PCR inhibitors; as well as phenol-chloroform-isoamyl alcohol extraction that is combined with additional chloroform:isoamyl alcohol (24:1) treatments (after the phenol-chloroform-isoamyl alcohol and before salt and isopropanol precipitation).
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Old 04-01-2008, 07:47 PM
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Default Re: PCR on "dirty" samples

First let me state that I work for Biomatrica. We have a new product that has been shown to amp product in the presence of inhibitors (300 year old bone samples buried in dirt). The product is calle PCRBoost and we have seen anywhere from 25% to 30 fold higher product when it has been used. it might help you in your application as well

Last edited by Bret Light; 04-01-2008 at 08:01 PM.
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Old 05-23-2008, 04:49 PM
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Default Re: PCR on "dirty" samples

If there are any template problems, I'd recommend using Qiagen's gel extraction / PCR purification spin column kit. Really makes a difference!
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Old 05-24-2008, 08:44 PM
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Default Re: PCR on "dirty" samples

Dear Elena

PCR is a very sensitive reaction and is often inhibited by certain compounds, which are co-purified with your DNA at the template prep step. Some include heme in blood DNA purification, humic acid in soil DNA purification and so on.
A number of papers expain methods of DNA purification from various samples claiming the PCRability of the template. However, you might need to standardize these methods and waste valuable time in doing so. The ideal thing would be, if your supervisor will spare you some cash, to get purification kits from QIAGEN or MObio. They are quality products and use cartridge-columns for purification, and are often worth the money especialy with regard to the time you would save by using them. If not, you can try different dilutions of the template, which could sufficiently bring down the concentration of the contaminant arbitrarily. And use different serial dilutions of the reaction for a reamplification step. Make sure its not a method-specific contaminant before you take any of these options. Phenol, often used to get rid of Protein contamination is a well known contaminant of PCR. a 70% wash is vital and often removes most of Phenol contamination.

Hope this was useful and you get your reaction right!
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Old 05-25-2008, 12:19 AM
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Default Re: PCR on "dirty" samples

Quote:
Originally Posted by ElenaKoulich View Post
Hi all!

I'm getting very tiny biopsy samples for my PCR analysis. I tried to purify DNA from these samples using conventional methods, adding glycogen, etc, but never got any detectable DNA to work with. I ended up incubating my samples in lysis buffer @95C for 10min, spinning them down, and using supernatant to run PCR. Sometimes this method works and I get PCR products, sometimes it does not. My guess is that I have contaminant(s) that inhibits PCR.
Does anybody have any good suggestions on how to clean up my samples?
Hi Elena,

My name is Bret Light and I am with a company called Biomatrica. WE have a product called PCRBoost that has shown to help PCR that has inhibitors in the reaction like that of 200 year old bone burried in soil. We were abe to get product just by the addition of PCRBoost to the reaction.

Sorrry for the commercial but it may help you with your research
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