Titanium Amplicon run.

[pmiguel]

Originally Posted by Anthony.287

Quick update on the AMPureXP test with denatured products – it doesn’t seem to work. My guess is that the AMPure and DNA don’t bind well, if at all, below a certain temperature. At the end of the process, the pools that were denatured retained very little, if any, DNA, while the controls still had plenty.
If anybody wants actual details, let me know! I’ll be happy to share.

I would be very interested to see your details. You actually attempted AMPure at an elevated temp? If temp is the issue, maybe you could use basic conditions (eg, 0.1N NaOH) to denature instead? Not sure if the beads would be stable in these conditions, though.

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Phillip

[Anthony.287]

Not quite. I denatured the DNA at 95C for 2 min, then 5min on ice. I added the AMPure while the sample was on ice (5 min) then took it off the ice and let it incubate at room temp for 10 minutes. I then proceeded with the AMPure protocol like normal. I also included controls of the same DNA that was not denatured. When I ran the samples on the Bioanalyzer when all was said and done, the controls had nice, strong peaks where they were expected, and the denatured samples had nothing. I repeated the test with new sample pools spiked with a 100bp ladder as an additional control, with the same results.

On a related note, does anyone have experience using the Roche Sizing Solution in place of the original AMPureXP buffer?

[pmiguel]

Originally Posted by Anthony.287

Not quite. I denatured the DNA at 95C for 2 min, then 5min on ice. I added the AMPure while the sample was on ice (5 min) then took it off the ice and let it incubate at room temp for 10 minutes. I then proceeded with the AMPure protocol like normal. I also included controls of the same DNA that was not denatured. When I ran the samples on the Bioanalyzer when all was said and done, the controls had nice, strong peaks where they were expected, and the denatured samples had nothing. I repeated the test with new sample pools spiked with a 100bp ladder as an additional control, with the same results.

On a related note, does anyone have experience using the Roche Sizing Solution in place of the original AMPureXP buffer?

How high did you go with your ratio of Ampure:sample? All the most ancient PEG ppt of DNA were done at 4 oC. (Okay, they were usually overnight precipitations, though.) Although the kinetics of the non-specific binding of DNA to carboxylated beads is not clear to me, doesn't seem likely that would be the cause.

Maybe it is your DNA being single stranded? That would mean half the molecular weight. My mental model for how a PEG precipitation happens is PEG occupying a percentage of the "solvation" sites sufficient to begin to force other molecules out of solution. Then the longer the molecule the more water molecules are needed to hold it in solution. But a single stranded molecule should take less water molecules because, well, only one strand to keep solvated. That would suggest it would take more PEG to precipitate single stranded molecules. Right?

The experiment I have not seen done is running the AmPure calibration curve on a single-stranded ladder. Maybe one could use the one included in the BioAnalyzer nano-chip kit?

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Phillip