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