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Consistancy issue with PCR block


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#1 Vyse

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Posted 20 August 2014 - 04:22 AM

I am having problems with my experiments on the 384 well block I am using, it seems inconsistant. When pippetting the exact same PCR mix (everything mixed into 1 tube) into random wells on the 384 plate, the result is variation in the fluorescence levels. However, the CPs dont seem to vary as much. There is no pattern as to which wells work better than others. One well might work great, then the well exactly next to it may fail to amplifly anything.

 

Is this likely a problem with my qPCR protocol conditions or is it a hard ware problem?

 

Many thanks



#2 Trof

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Posted 20 August 2014 - 08:22 AM

At first it woudn't hurt to know what cycler do you have and all the other details (like, was it consistent before, what amount do you pipett, how do you pipett it, what system are you using - SYBR/Taqman,...).

 

Also, "Cp doesn't seem to vary much" and "well next to it fail to amplify anything" seems kind of contradictory to me. So what exactly is happening? What do you call  "fluorescense" (the Cp is what you get after analysis, that doesn't need to be specified I hope).


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