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PCR efficiency in real timePCR


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5 replies to this topic

#1 pop09

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Posted 11 February 2010 - 11:18 AM

Hi, I cannot quite explain why, with the same DNA stock sample, I am getting >90% PCR efficiency in my realtime PCR runs using 2-fold dilutions of standard samples, but <80% efficiency using 10-fold dilution series.

Help!

#2 jpopesku

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Posted 16 February 2010 - 12:02 PM

Hi pop09,
That is indeed strange, as the dynamic range of real-time is quite large.
I suspect that the problem you're experiencing is related to the serial dilutions: if you are doing a 2-fold dilution, you are transferring 50% of your sample to the next dilution at a time, whereas with a 10-fold dilution, only 10% is being transferred. Thus mixing might be the problem.
Make sure that the tube is very well mixed (i.e. vortexed or pipetted up/down >40 times) after each transfer....

#3 stylothecancer

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Posted 16 February 2010 - 10:25 PM

Hi, I cannot quite explain why, with the same DNA stock sample, I am getting >90% PCR efficiency in my realtime PCR runs using 2-fold dilutions of standard samples, but <80% efficiency using 10-fold dilution series.

Help!


hI pop09,

Have you check on the sensitivity level of your assay as well as the saturation point of your assay?

#4 Baars01

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Posted 17 February 2010 - 03:55 AM

Do you use more 10-fold sample in your PCR rxn than 2-fold sample?

#5 tea-test

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Posted 17 February 2010 - 11:04 AM

are the two std curves over the same dilution range? If not, I assume the range of the 2-fold dilution is much smaller (like 1:100) and this is not very accurate. I have attached some literature where the difference is explained.

Attached Files


tea-test: The artist formerly known as Ned Land

#6 pop09

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Posted 19 February 2010 - 09:22 PM

Hi all thank you for your comments. I will check out the article...What do the sensitivity and saturation point mean? Obviously I am a novice to this.




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