Protocol Online logo
Top : New Forum Archives (2009-): : PCR, RT-PCR and Real-Time PCR

qPCR volume - Problem with volume difference and results (Apr/07/2011 )


We are working with LC 480 and doing 1 step qPCR.
we are working with 10無, if the settings of the PCR was set to 20無 instead of 10無 like we did, is it a problem?

Our problem is that we did the standard curves (with 10無) and measured 3 plates already (with 10無) but forgot to change the setting of the PCR run from 20無 to 10無, and when we changed it with our fourth plate we had a difference of 7 cp values for the target gene and no difference in regard to the reference gene!

Does this difference has to do with the settings of the LightCycler or is it a normal result that has to do with the samples (taking into consideration that the Standard and the Calibrator of the target gene was also increased by 7 cp values)?



From what I heard from Roche representative regarding this question, the volume setting only affect the area in which fluorescence is read, if you set 20ul it will make a wider circle to read fluorescence from, and if you lower the volume, the circle is appropriately reduced. So if you set a 20ul and really have only 10, it just reads wider circle than necessary, which may affect a bit the level of the background fluorescence, but no other things.
It's hard to say what caused your gene to jump 7 Cts.