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Waht can we do??! Non specific bands in PCR with dog blood samples.

Waht can we do??! Non specific bands in PCR with dog blood samples. - PCR - Polymerase Chain Reaction Forum

Waht can we do??! Non specific bands in PCR with dog blood 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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Old 07-01-2010, 08:23 PM
Pipette Filler
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Unhappy Waht can we do??! Non specific bands in PCR with dog blood samples.



Hey there, I 'm posting a question for a friend of mine here at the lab.
He's running PCR for detection of Trypanosoma cruzi, from blood samples of infected dogs. The thing is he gets a lot of smear, non specific bands I mean. He also sometimes gets the band he' s looking for. But it's really hard to know if it is a consistent result, because of all of the other bands he keeps geting.
He told me that he tried lowering the Mg and it was worst! Also, when he adds a larger sample in the PCR, he gets a more distinct band for T. cruzi but also more non specific bands.
We think that, evidently, all of the bands he's seeing, come from dog's DNA. But how could he prevent this? Or how could he enlarge the T. cruzi DNA in his samples..?

What dou you think he could do?

Thanks so much!

cheers
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Old 07-14-2010, 06:12 AM
Pipette Filler
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Default Re: Waht can we do??! Non specific bands in PCR with dog blood samples.

There are many reasons for getting non specific product/s smear. First of all It would be helpful to know if this pcr has been done before and worked it ? Is the used annealing temperature correct ? Try to higher it in 0,5-2 C increments. What about the primers, are they self designed ? Maybe it is a specifity Problem. Try to check with Primer Blast on NCBI. What about your enzyme ? Is it still intact ? Try to change. Is the cycle count to high ? Enzyme amplifies unspecific when cycle number is too excessive. Try to lower.
Enzyme presence at very high concentrations may cause unspecific amplification, too. Generally use enzyme concentrations at 0,02-0,05 U/l (dependend on enzyme used and amplicon length).
Is the Primer concentration to high ? Very high primer concentrations may effect unspecific binding. What about your negative control. Is that okay ? If not, it is a contamination problem. Is the DNA sample possibly broken (long storage) into fragments or is the purity of the sample given as needed (protein contamination, Dnase). Is the template of high GC% content? Initial melting at higher temperature (first 3 steps at 98C 5-10s) and adding of DMSO (5-10%) should help.
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