Disagreement between triplicate results in Q -PCR
Posted 08 September 2011 - 07:45 PM
I am new to Q-PCR.
Recently, i have been using SYBgreen Q-PCR to check the relative gene expression . For every samples i did it in triplicate, however, i always get a disaggrement or large variations between the results of my triplicate trials including a difference of Ct value and a non-smooth curve in the amplication plot within a sample with triplicate trials.
I was just wondering how i can improve the smoothness of the amplication plot of those triplicate trials of a sample and what tricks you guys are using in order to have a nice Q-PCR result. I am sure that i have pipetted an equal volume of mastermix into each sample in the eppendorf tube and then into the 96 well plate. And i am sure that i have mixed well the solution by pipetting up and down for about 7 times. Yet, i still get a bad result....
Pls kindly give me some suggestions or tips..
Posted 09 September 2011 - 07:23 PM
Posted 10 September 2011 - 12:27 AM
Well i have not checked the accuracy, um.. i dun know but i think it's ok
Posted 10 September 2011 - 06:20 PM
What kinds of differences in Ct are you getting between triplicates?
Posted 11 September 2011 - 03:45 PM
Posted 13 September 2011 - 12:34 AM
Difference of 1 to 2 cycles in what exact Ct value? It's normal to get such high variation in later cycles (like 33) but certainly not around say 20.
The Ct Difference I get is about 1 to 2 cycles. Is it too large?
You say you added 10 ul of sample to the reaction of what final volume? If it is a cDNA in RT mixture, recommended maximum sample volume is 10% of the final volume. So unless you have a 100ul reaction, that's way too much.
Edited by Trof, 13 September 2011 - 03:47 AM.
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