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high Cp value PCR


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#1 susanna

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Posted 08 October 2009 - 07:06 AM

Hi,

In our lab, we are working with qPCR, for mutation detection. Now the problem is that all of our Cp-values, for our test DNA, are too high. The values are over Cp: 32 and more, for our test DNA. We don't know what the problem is, since we used purified DNA (so it can't be that its because of contamination, I think).
Because of this, we don't dear to start, with mutationdection of our samples.
Do somebody now, what kind of problem can cause this effect? This would certainly help,

thanks,

Susan

#2 Flyingbike

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Posted 12 October 2009 - 05:03 AM

Hi,

In our lab, we are working with qPCR, for mutation detection. Now the problem is that all of our Cp-values, for our test DNA, are too high. The values are over Cp: 32 and more, for our test DNA. We don't know what the problem is, since we used purified DNA (so it can't be that its because of contamination, I think).
Because of this, we don't dear to start, with mutationdection of our samples.
Do somebody now, what kind of problem can cause this effect? This would certainly help,

thanks,

Susan



I think this means a low efficiency PCR. Are your parameters (temperature, elongation time, etc) adjusted according to your amplification target ?

#3 susanna

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Posted 15 October 2009 - 04:06 AM

Yes, but i wondered why i have such a low pcr-efficiency.
Were using a kit, so everythings already optimalized.

greetz

susan


Hi,

In our lab, we are working with qPCR, for mutation detection. Now the problem is that all of our Cp-values, for our test DNA, are too high. The values are over Cp: 32 and more, for our test DNA. We don't know what the problem is, since we used purified DNA (so it can't be that its because of contamination, I think).
Because of this, we don't dear to start, with mutationdection of our samples.
Do somebody now, what kind of problem can cause this effect? This would certainly help,

thanks,

Susan



I think this means a low efficiency PCR. Are your parameters (temperature, elongation time, etc) adjusted according to your amplification target ?






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