5 replies to this topic

[pop09]

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!

[jpopesku]

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....

[stylothecancer]

pop09, on Feb 12 2010, 03:18 AM, said:

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?

[Baars01]

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

[tea-test]

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

•  Application_note_Understanding_Ct.pdf   2.03MB   72 downloads

[pop09]

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.