Standardizing cDNA concentration before doing PCR - (May/05/2015 )
I was told that I should standardize my cDNA concentration before going onto PCR, so as to ensure consistent results.
I have not done this so far, and my PCR results have been pretty good. But should I as a good habit/scientific practice?
I don't think this is necessary. Often cDNA concentrations are quite low, but that is fine for use as a PCR template.
I know this is late, but thank you for your reply!
I have recently noticed that I am getting very faint bands when my RNA concentration before RT is high (250-350 ng/uL). Otherwise, the PCR bands are pretty good when my RNA concentration before RT is about 100ng/uL. In the future, should I dilute the RNA to around 100ng/uL before RT to ensure good cDNA synthesis and PCR?
Extracted DNA and RNA often has ontaminants that inhibit reactions. Often, diluting before reactions. or adding smaller amounts, will make a reaction succeed which would otherwise fail. Many or most reactions need smaller amounts of template than expected.
I have heard the same thing from our lab tech, thanks!
It's funny, my RNA extraction is suddenly yielding quite low concentration of RNA, going from ~100-200ng/uL to ~30-60ng/uL. I have not done anything different, and am using freshly autoclaved tubes and tips. It could be that I am extracting RNA at a different time point, where gene expression may be low, but I didn't even get this low with my naive animals... Any ideas why?