cDNA dilution - to dilute or not? (Apr/21/2008 )

Hi,
I was wondering if anyone using cDNA in a pcr reaction normally diluted thier cDNA, and if so by what dilution and what kind of buffer would you use? If anyone had a recipe that would be brilliant! also has anyone ever used neat cDNA in a pcr i.e the product from a superscript II reaction without diluting it?
thank u!

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Yes, I sometimes run 1.0-1.3 µl of the neat cDNA in a 50µl PCR reaction if the transcript seems difficult to amplify -- but I usually start with 2.5 µl of a 1:5 dilution of the cDNA. (Diluted in water). Hope this helps.

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Thanks a lot thats a big help! Im assuming u use nuclease free water?

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You're welcome -- yes, I use autoclaved Milli-Q water for PCR (but I always use DEPC treated water for the RT reactions.)

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The cDNA synthesis usually wields 20uL of cDNA. I dilute it with an aditional 40uL of DEPC-treated water and use 2uL in each pcr reaction.

It works just fine, you just may need to pcr for some extra cycles to get a good result.

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Thanks guys, i used nuclease free water to dilute and it worked a treat!

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