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Old 03-12-2011, 09:33 AM   #14
Location: Washington State

Join Date: Mar 2010
Posts: 12

I would be shocked if ABI didn't jump on the automation problem before the transaction was complete. As for posting, I usually don't. I can't post too much because I really enjoy being this old and still having "junior member" status. But at this moment I feel I can contibute to anyone thinking about jumping in, or getting started w/emPCR for the first time.

Ideally, corners to cut in any proceedure are those that make it faster and better. For starters I washed the plate more, because I'm greedy, then stacked the aqueous in fewer tubes for less handling. I nearly eliminated the spins at both the non-polar and polar extractions. A DNA covered bead has so much charge that is isn't going to be anywhere near hexane. A hard 5' spin to separate the phases is unneccesary, we aren't trying to pellet the beads here. Anything more than a long pulse only puts junk you want to get rid of closer to the interface. There won't be anything you want at the interface, so it is OK to lose a ul or two of aqueous, to get the sample cleaner, quicker with fewer extractions.

The tiny beads are almost invisible, but seem to make a dense enough pellet with shorter spins. I was able to do more thorough washes by getting closer to the pellet when removing the super. It felt dangerous not spining long and getting all the super out, and it was, but it worked out fine. I melted the enriched beads off with 2x5' alkali soaks, instead of 1x15', etc. etc. etc.

The real fine line that needs to be walked, as with the 454, is the ratios going into emPCR. Only good quantification will help here. Aim low for fewer higher quality beads, then get greedy during their purification. Ideally you want to harvest just enough beads to proceed. All common sense stuff, wish it was worth more than $0.02, but it is all obvious.
wildung is offline   Reply With Quote