PCR after in vitro transcription and translation - (Aug/24/2006 )

I'm running an in vitro transcription and translation reaction using Roche's RTS E. coli HY kit. I put in a linear DNA template, successfully make protein, and then I attempt to recover the DNA by PCR. Nothing is coming up in the PCR though. I know my PCR conditions are fine because I have a positive control of the DNA going into the same volume of water as the RTS reaction and then that shows up in the PCR.

One thought is that the DNA is being degraded in the reaction. I've tried adding salmon sperm DNA to the reaction hoping to slow any degradation but that doesn't help. Another possibility could be that some RTS components are interfering with the PCR reaction. This seems unlikely though, because I've added as little as 0.06 ul to a 40 ul PCR reaction (by diluting of course) but that still works out to 0.24 ng of starting DNA which should be enough for PCR I assume. I've run up to 35 cycles.

Any idea what is happening here?

Hi,

Not sure I understand what's going on here... so you're trying to amplify the template that you used for the in vitro transcription/translation after the reaction has proceeded?

Depending on the amount of template you're initially using, I suppose you could add some type of carrier protein or PEG and PCI/EtOH extract the DNA, then try using that as a template for your PCR.

_Hank

Correct, I'm amplifying the DNA from the IVT reaction after it's done. I know this sounds kinda weird but it's because I'm trying to do a selection similar to Sepp, et al. (2002) FEBS Letters 532, 455. Right now I'm using between 50-200 ng of DNA in a 50 ul IVT reaction, but eventually I'm hoping to get it working with ~1 ng. I don't really have much experience with PCI/EtOH extractions but I'm guessing below 1 ng of DNA would be hard to extract?