4 replies to this topic

[dac63]

I'm wondering if anyone out there has had a similar experience:

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
extractions.

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
contaminating proteins.

Any suggestions/ideas would be greatly appreciated!

DC

[promilasharma]

Actually the aqeous phase you are taking is too small.If your DNA is dissolved in  small volume of water ,first you dilute it  with sterile water to make up the volume minimum 200 ul  and then do chloroform:isoamyl alcohol treatment in 1:1 ratio .Once you have separated the aqeous phase then reprecipitate it  and finally dissolve the pellet in minimum volume of water.

[dac63]

Thanks for the suggestion. I'm just following the directions
in the  Clontech cDNA Subtraction Kit, but if I keep losing
valuable mRNA doing it their way then it's time for Plan B.

DC

[LabPenguin]

Have you tried this:

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.

[sarah]

I found that using phaselock tubes from eppendorf solved all my problems of removing the aqeous phase, no matter how small. They lock the organic phase below a gel laye, making it possible to simply decant the aqeous phase off. Well worth a look.