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[byobortolazzo]

Hey guys,

So prior to today, I've only been using the Invitrogen Miniprep kit. Using that protocol I start with ~5-6mL bacterial culture and spin the WHOLE thing down and work with that. I'm missing one or two things to proceed with this kit anymore, so now I'm movinng on to our Promega PureYield kit.

This one, however initially says to start with 600uL of culture, and that alternatively, 3mL of culture can be used for higher yields.

Being that I was "nestled" on always using 6mL cultures completely, I'm not sure what I should do. Does anybody here have experience of using more culture than what these protocols specify for? I want as much DNA as possible, but I don't want to run two collomns for each culture, really.

Thanks!

Edited by byobortolazzo, 18 February 2012 - 12:32 PM.

[phage434]

Two things limit the amount:
1) The lysis of cells may not be complete
2) The columns may not bind all of the released DNA

1) is a serious problem, and you cannot get good DNA without good lysis.  If necessary, add more lysis reagent (and more neutralization reagent).  You probably want to use less culture next time.  You can load the column in several steps, if necessary due to higher volumes.

2) is not a problem, but there is no point in lysing a lot of cells with no increase in yield.

In general, they will tell you the volume that works best.