conc of cDNA to be used - PCR to test primers (Apr/21/2008 )

Hi I have primers to be tested on my cDNAs. Was wondering if you use diluted cDNA(1:20) or just concentrated cDNA.
I started with 1ug RNA in each sample. Thanks!

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I always used 1ul of undiluted, 25 cycles for my first PCRs with a housekeeping gene. And then I'd adjust if required after that. But I think people have many different ways in which they do it

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i think 1 ug of RNA could be a lot, however it depends on the protocol you're using to sinthesize cDNA. i generally used 500 ng of RNA for a final volume of 25 ul of cDNA. then 1 ul of undiluted cDNA in a 50 ul pcr rxn.

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You can use up to 5µg RNA in an RT reaction, but I usually use 2µg (in a 20µl RT reaction). For PCR, I usually dilute the cDNA 1:5 in water and use 2.5µl in a 50µl reaction. Alternately, if the transcript is low abundance or difficult to amplify, I would use 1.0-1.3µl of undiluted RT in the 50µl PCR reaction.

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For me: 250ng up to 1ug RNA in 20ul RT reaction, for PCR 2ul of 1/10 dilution (in nuclease free water) in a 12 or 20ul total volume.

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correction: the final volume i use is not 25 but 20 ul of cDNA. you should consider there may be small traces of genomic DNA in your initial RNA, so, the more RNA you use for your cDNA the higher the possibility of increasing your DNA contamination.
cheers,
toejam

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