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Chip pcr. are there inespecifics?

chip pcr

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#1 valeri_a

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Posted 06 June 2013 - 07:41 AM

Hi guys!.
After IP and reverse cross linking and DNA purification, I done a standard PCR using control primers (GAPDH), wich I aspect bands around 166 bp.

But.. This is my result of my standard PCR the samples are attached:

1- positive control (RNA pol)
2- negative control ( IgG)
3- intesest target (HA)
4- Input
5- negative control of reaction (water)



I don´t found explanation for this results. probably I have product in the imput, but I guess that I don´t have nothing what I looking for. And what about the amount of fragment of the IP product? is amazing. Is that because the IP is failed?, or it could be the pcr condictions??


another question , it could be that products forms dimeric structure??. or are those inespecific products?

thanks for your point of view!!!!

cheers,
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#2 jerryshelly1

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Posted 06 June 2013 - 09:55 AM

You used GAPDH primers for all of your samples? Did you try these primers on a sample that you know should show amplification? It appears as if you have formation of some kind of product, just not at the expected size. I would test these primers and see if they are still viable. You may have some possible degradation occuring in your samples or inaccurate PCR conditions. It is also important to note the amount of smearing that is in each lane. This can be indicative of low annealing temperatures, overabundance of template, etc... Good luck.





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