Chloroform-isoamyl extraction problem
Posted 12 January 2005 - 10:26 PM
When I try to removed the aqueous phase (40 ul) from a chloroform-
isoamyl alchol (24:1) extraction of a DNA solution, I am only able to
remove a small fraction of the aqueous phase before it forms a small
bubble surrounded by the organic phase, i.e., the meniscus disappears.
After centrifugation, the aqueous phase is on top as expected. This
also happens to a much lesser extent with my phenol:chloroform:isoamyl
I'm using a 1:1 ratio of aqueous and organic phases, but could the salt
concentration be too high in the aqueous phase, for example? Or could it
be a problem with the CI prep? Besides trying (with limited success) to catch
a tiny aqueous bubble with the pipet tip, obviously this also transfers
Any suggestions/ideas would be greatly appreciated!
Posted 13 January 2005 - 01:24 AM
Posted 14 January 2005 - 12:17 AM
in the Clontech cDNA Subtraction Kit, but if I keep losing
valuable mRNA doing it their way then it's time for Plan B.
Posted 28 January 2005 - 02:02 AM
I pipette off the organic layer. Although you lose a tiny bit of the aqueous layer at the end, in making sure that you don't leave any chloroform behind, this has the added advantage of reducing shearing of the DNA which would otherwise ocur when you suck the aqueous layer into the pipette tip.
Posted 08 February 2005 - 03:32 AM