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high Cp value PCR - (Oct/08/2009 )

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

-susanna-

susanna on Oct 8 2009, 05:06 PM said:

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 ?

-Flyingbike-

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

greetz

susan


Flyingbike on Oct 12 2009, 06:03 AM said:

susanna on Oct 8 2009, 05:06 PM said:

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 ?

-susanna-