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PCR Real Time Well to well variation

PCR Real Time Well to well variation - PCR - Polymerase Chain Reaction Forum

PCR Real Time Well to well variation - PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory.


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Old 08-21-2012, 06:56 PM
Pipette Filler
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Default PCR Real Time Well to well variation



Hi everybody. Since a few months I have seen a lot of variations between my triplicates. And I do not know the cause of that high variation.
When I perform my Real Time PCR i prepare a Mx without Rox and then dispense 16 uL of the Mx in each well and then dispense 4 uL of each sample for triplicate.
My ct values are in the 22-29 range. And sometimes I obtained a difference about 3 or 4 between the same sample (I mean, for example for the sample called 4651-2 the ct values give me: 26.56, 27.03 and 23.25) and it is a really mess.
I have seen many times that when I dispense the 16 uL of Master Mix or the 4 uL of each sample it kept a little drop in the tip. But I have been reading about pcr-real time troubleshootings and many people say that this little drop it cannot be the cause of such high difference between triplicates ct. And that is because a difference of only one in the ct it means you have double of copies (double volume) between wells. So that cannot be the cause.
My question is: What are the causes about the differences between my cts in triplicates??
Another thing i have to say is that differences between cts are randomly.
I will really be greeting if someone can help me to solve my problem.
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