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-   -   With 40 pcr cycles, how relevant are samples with Ct's 35-40? (http://www.molecularstation.com/forum/real-time-pcr-quantitative-pcr-forum/72580-40-pcr-cycles-how-relevant-samples-cts-35-40-a.html)

wozzels 03-10-2010 09:07 AM

With 40 pcr cycles, how relevant are samples with Ct's 35-40?
 
Hi,

If i run 40 pcr cycles in a real-time pcr experiment, are Ct-values up to 40 relevant and meaningful, or should i discard results having a Ct of more than 35? And do blank samples have Ct-values equal to the max number of cycles, or not? Because if they do, unknown samples with high Ct-values may indicate the absence of DNA template right?
This is a question that is bothering me for quite some time now because I don't want to reject possible positive results but I also do not want to draw conclusions about samples which may not even contain my gene of interest.

Thank you guys in advance!
Wozzels

danfive 03-10-2010 07:06 PM

Re: With 40 pcr cycles, how relevant are samples with Ct's 35-40?
 
Quote:

Originally Posted by wozzels (Post 419271)
Hi,

If i run 40 pcr cycles in a real-time pcr experiment, are Ct-values up to 40 relevant and meaningful, or should i discard results having a Ct of more than 35? And do blank samples have Ct-values equal to the max number of cycles, or not? Because if they do, unknown samples with high Ct-values may indicate the absence of DNA template right?
This is a question that is bothering me for quite some time now because I don't want to reject possible positive results but I also do not want to draw conclusions about samples which may not even contain my gene of interest.

Thank you guys in advance!
Wozzels

Depends on the nature of your gene and the quality of the DNA, and the efficiency/quality of the PCR assay (mostly primer + amplicon dependent)

Assuming excellent quality DNA (not shredded, digested, no PCR inhibitors present).

An amplification at 40 or more cycles is still relevant
due to any of the fllwng:
*low dna template copies
*high gc region, difficult to amplify/melt apart
*low binding efficiency of primers (primer sequence dependent)
*un-optimized PCR conditions, thermocycle program, reagent conc'ns

Some PCR assays literally use nested, semi-nested (totalling ~80 cycles) because of low expression/weak amplification in the first round.

Blanks should be blank no matter what, in real time assays the flourescent noise sometimes creeps up, but that should be rare and should not resemble a weak positive amplification (should look like noise, if that is not the case suspect contamination).

Since I don't know what you're doing, note that:

In diagnostic PCR: confirm the dubious samples with different primers, regular PCR, extra starting template etc.

In knock-out cloning: breed sample into next generation to confirm absence/presence of gene-of-interest.

muffinman563 05-11-2010 08:39 PM

Re: With 40 pcr cycles, how relevant are samples with Ct's 35-40?
 
You can try to make a different concentration (higher) of the target sequence to decrease Ct value. That may work.


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