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qPCR please help

qPCR please help - PCR - Polymerase Chain Reaction Forum

qPCR please help - 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 11-06-2009, 03:54 PM
Pipette Filler
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Unhappy qPCR please help



Heya,

I am having problems with my qPCR. I am trying to amplify the humand ADH1A gene. I have primers (do not know the sequence) that seem to work fine amplyfing the gene and they give me a nice melting curve. However, my negative control (primers only) comes up positive:-( I have repeated the experiment so many times now it is really frustrating. I have been really careful handeling the plate, samples, and reagents. I have used new water and primers.

Could it be that the ADH1A gene is structurally difficult to amplify??
I really appreciate any comments.

Thanks for your help.
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  #2  
Old 11-07-2009, 12:32 AM
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Default Re: qPCR please help

From what you've said it seems that ADH1A gene in your reactions amplifies just fine and the problem is your negative control.
What kind of qPCR do you perform? Is it based on fluorescent probes (like hybrydization probes that can be used with melting curve analysis)/SYBR Green I/other assay? Do you use any commercial kit for ADH1A or do you prepare your assay on your own (primers, polymerase mix etc.)?
If your negative control seems to be positive then what kind of positive signal do you obtain for it? (This actually is based on the kind of assay you perform - whether it is unspecific probe/probe degradation or primer dimers in SYBR Green I based assay.) What is Cp for your negative control?
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  #3  
Old 11-09-2009, 09:18 AM
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Default Re: qPCR please help

Dear Aga,

thank you for your reply.

My qPCR is based on SYBR green assay (Roche) and the primers I am using have been optimized already. I have ordered these primers and the company has sent me a quality form along with them. It shows a single dissociated curve peak around 82C. So I would think that the primers work in qPCR. Previously I had used my own primers but I had a similar problem with the negative control coming up. So I decided to go with optimized primers.

For my negative control I also get a Dissociation curve peak at 82C. When I analyse my samples my negative control looks like my positive with a 'nice' single peak at the same temperature.

I do not think that I am getting primer dimers as I believe that if they were dimers then I would expect a peak at a lower temperature (maybe 70C).

I have run the negative control sample on a gel and bands were visible at around 150bp (the ADH1A fragment I am trying to amplify is about 148bp).

I am not sure what to do anymore. I have ordered a new set of primers as the only other thing I can think of is contamination:-( I have set up the qPCR in different rooms/hoods already and even other people have set up a reaction for me already and still the negative comes up positive.

Thanks for your help, any suggestions are very welcome.
Julia
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