500 ng RNA is converted to cDNA to be genetically expressed by qpcr sybergreen dye, 1 ul primer F, 1 ul primer R, 10 ul master mix, 1 ul cdna template, and instead of a complete total volume reaction to 20 ul, by mistake I added 13 ul water instead of 7 ul. so reaction volume becomes 26ul in each well.
is it a fetal mistake? Can I readjust it. is it okay to go on in reaction?
should not a problem as long as you prep any experiments to compare to this in a similar fashion.
you should re-check the efficiency with a serial diluted standard in a reaction prepared the same way, it is it close to what the efficiency is with it prepared normally (within 5%) then it is ok.
I'd tell you to run the reaction and see what the data say.
What did you observe?
As Julie Ann Dougherty says it probably won't make much difference. Nevertheless since you have to use the same conditions for all the qPCRs to compare, I'd rather repeat this one with the correct quantities.
I did the run. And i found that some result is rationable to the previous one.
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