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-   -   same primers, different products in conv PCR and qPCR (http://www.molecularstation.com/forum/pcr-polymerase-chain-reaction-forum/86212-same-primers-different-products-conv-pcr-qpcr.html)

alqga 08-14-2012 08:18 PM

same primers, different products in conv PCR and qPCR
 
Hello,

I am new here and I donīt know if this question has already been answered but here it goes.

I have run a qPCR with new primers and I got amplification and nice melting curves. IN addition I loaded an agarose gel and the product had the expected size (around 180 bp)

However, I wanted to sequence the product to be sure and when I run the conventional PCR prior to the purification of the DNA and I loaded a gel to see the size of the product I obtain a huuuge product (1000 bp) what has nothing to do with the product I got in the qPCR

Any hint about what can be?? (sorry if this is too basic but Iīm quite new...)

Thanks!

jonoave 08-19-2012 03:30 PM

Re: same primers, different products in conv PCR and qPCR
 
There could be many factors:
1. The reagents you used for your PCR and qPCR are different.
2. Different cycling parameters and temperatures between PCR and qPCR.
3. Less likely factor, different machines used between PCR and qPCR.

PCR is a very sensitive process, and variations can lead to erm, variable products.
I'm not really what you're asking - if you need your qPCR to work I guess you should try to make the cycling parameters as close as possible to regular PCR?

germagno86 08-22-2012 05:27 PM

Re: same primers, different products in conv PCR and qPCR
 
I suspect you got a contamination in conventional pcr in one of your reactives when you prepaired the master mix.


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