I am using Applied Biosystems 7500 Fast system for qPCR. I am using 10ul sample per well. Today I run the sample, however, I forgot to change the sample volume to 10ul from 20ul in the software protocol. So will this matter in my analysis?
I guess it will, if you didn't change the sample volume in the software settings, all calculations were made as for 20uL sample. But it depends on what do you want to analyze.
I think it's not a big deal. If you want to do some quantification, just keep in mind that your values are calculated for a 20uL sample. I am not familiar with the software from AB, but sometimes it is possible to change settings offline, after the experiment ,and all results are then simply recalculated.
The issue could be that the temperature during your PCR was not optimal as the machine takes into account the volume of your sample to control precisely the temp in your well. If you are comparing your data with others from different run, then your results could be affected. I would be curious to know if it makes a big difference at the end though. If all you do is to compare (samples /std curve..) from the same run then I don't think it will make a big difference. I would be curious to know!
as long as all your analysis is dependent only on this plate, you are fine. your standards have been input as copies, i believe. all your unknowns will still be based on the Rf values as per your actual copy number of the standards.
hey guys, thanks for the suggestions. There was no error in experiment. It didn't make any difference to std curve assay.
I was wondering if anyone uses the Bio-Rad CFX96 Real Time system and have encountered the same problem?
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