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problem with ChiP q-PCR

qPCR ChiP primers gc-ric

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#1 giuly984

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Posted 21 November 2013 - 08:53 AM

Dear all, 

I have a dated and quite annoying problem with ChiP q-PCR primers. 

I have to check the binding of a transcription factor on the murine cyclin E1 promoter. I identified 2 hypothetical binding sites with the help of the matinspector software from genomatix, and tried to design primers to amplify the regions of interest.

Like many other promoter regions, unfortunately, the cyce1 promoter is quite rich in GC, and this aspect probably increased the difficulties I have.

I designed multiple primer pairs with primer3 and tested them on a gradient and after that for efficiency, on a input dilution scale. These are the main problems I encountered:

-on a gradient (54-64 °C), the curves ct are very distant and the melting curves are not unique (differ from temp to temp)

-surprisingly (considering the gc%), low temperatures show better melting curves than higher

-I identified 2 pairs with an acceptable efficiency, but:

     -in one case at low concentration the melting was terrible (and I have low concentrations of some of the samples to be tested!)

     - in the other case the melting was perfect, but the primers appeared to have a "low sensitivity", in the sense that the 1st dilution appeared 5 ct later than the same sample compared to a reference primer pair. (and due to the fact that I have low concentration in my samples, I cannot detect any amplicon in these conditions in my samples!)

 

I am really annoyed and under pressure,

and I have to solve these problems soon.

I am using sybr green, and I tried different optimized mix.

Thank you in advance,

Giuliana



#2 phage434

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Posted 21 November 2013 - 10:28 AM

Try adding 3-6% of a 1M betaine solution to your PCR reactions.

 

Can you do a control PCR reaction from genomic DNA rather than from a CHIP sample?



#3 giuly984

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Posted 28 November 2013 - 02:35 AM

Thank you!

I received a sample product of betaine today. would it function also for Real Time per? or should I perform classic PCR?

thanks



#4 phage434

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Posted 28 November 2013 - 06:55 AM

You should be able to add it to a qPCR reaction without difficulty. If you are doing quantitation, make sure you add it as a master mix to all samples equally.



#5 giuly984

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Posted 28 November 2013 - 07:50 AM

i'll try asap!






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