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Long-amplicon QPCR of mtDNA


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#1 janap

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Posted 07 February 2011 - 06:31 AM

Hi everybody. I really need your help, because I am stuck with my QPCR work for several months. I am using long amplicon quantitative PCR, often called simply as QPCR (Santos et al., 2006) to detect mtDNA damage in C2C12 cells. The method requires PCR to be kept in the exponential phase because the reaction must be quantitative, it means that amplification yields must be directly proportional to the starting amount of template.
I optimized conditions for the long amplicon (~ 10 kb) and I already got good results which means I got the right product on agarose gel and with 50% reduction of template DNA I got 50% reduction of amplification signal. Unfortunatelly it was not repeatable for the next time and for large amount of samples. Then I started to use AmpliWax Hot start and Genomic-tips for isolation of DNA (instead of Dneasy Kit I used before) because it should isolate high molecular weight DNA and improve the results of long PCR but I got no product in my PCR with this DNA. Does anybody have experience with this type of PCR? I will be happy for any idea!

Thank you in advance.

Jana

#2 Maddie

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Posted 07 February 2011 - 11:09 PM

Hi Jana,

There could be several reasons. The first that comes to mind is inhibition because you use too much DNA. Have you tried diluting your extract?
Another possibility is if your DNA degraded and you don't have so many intact 10kb templates anymore. I would start with the dilution test and then eventually you could run some of your extract on an agarose gel to see if you have DNA and what size it is.Good luck.
Theory is when we know everything and nothing is working. Practice is when everything is working and nobody knows why. Here, we combine theory and practice. Nothing is working and nobody knows why.

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#3 janap

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Posted 09 February 2011 - 05:31 AM

Thanks for tips! Anyway, I don't think the problem is in too much template DNA as I've already tried dilutions. I am now pretty sure, that the problem is in the quality of DNA. I've ran my template DNA on 0.5% agarose gel and got strange results. The gel itself isn't very nice and there is too much DNA loaded, but what I am not sure about are the short bands? Could it be RNA? I am not using RNase treatment because I read that RNA shouldn't interfere with PCR reaction.. I use Genomic-tips for DNA isolation and purification and follow the protocol for tissues as I need to isolate in particular mtDNA. Any experiences or tips as for this protocol??Jana

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Edited by janap, 09 February 2011 - 05:42 AM.


#4 Maddie

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Posted 12 February 2011 - 07:16 PM

Ethidium bromide intercaltes between the 2 strands of dsDNA so I doubt this is RNA. But I don't work with RNA so, I can't guarantee anything. It just doesn't seem plausible to me.
Is this gel showing your extract? What size are the small bands?
Theory is when we know everything and nothing is working. Practice is when everything is working and nobody knows why. Here, we combine theory and practice. Nothing is working and nobody knows why.

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#5 janap

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Posted 14 February 2011 - 01:56 AM

Hi, I also don't work with RNA , it was just such an idea, probably I am wrong.. There is 10 ul of extracted DNA loaded on the gel and there is only cell lysate in some wells (2 and 6) and wash fraction (3 and 4), the rest is gDNA. I used HindIII digest of lambda phage DNA as a marker, so the short visible bands are 2.3 and 2 kb in length. I think that the small bands in my samples could be around 1.5 and 1 kb in length, so it really won't be RNA..
Jana

#6 Trof

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Posted 17 February 2011 - 01:30 AM

Ethidium bromide intercaltes between the 2 strands of dsDNA so I doubt this is RNA. But I don't work with RNA so, I can't guarantee anything.

Just note: running denatured RNA (in formamide) you can obtain visible bands on gel with EtBr, it's commonly used for testing RNA quality. I don't understand the principle either, but RNA can by visualized with EtBr at certain conditions.

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