DNA cleanup - (Apr/12/2008 )
I need to cleanup DNA constructs for use in fly embryo injection. I was told by lab members that I need to phenol-chloroform cleanup the DNA after maxi/mini preps because apparently the columns introduces something that messes with the embryo development. The inital DNA that I have was maxi-preped and is at around 300ng/ul. With 60/80 of 1.8. So its pretty good.
I took 100ul of the construct and I phenol-chloroformed the DNA, ethanol precipiated it, reconstituted it in ddH2O. The DNA was concentrated to about 500-600ng/ul. So that was good, but the 60/80 went down to 1.5. I figure maybe some reagent in the ethanol precipitation got contaminated. I switched out for fresh ethanol, fresh NaoAC and H2O. The OD is still 1.5. What am I doing wrong? I've already phenol-chloroformed it 3 times now.
Make sure you are eliminating all the phenol. Try either an extra chloroform only extract (or more than one), or an ether extraction. Be careful with the ether -- it's both very flammable and, if dried, potentially explosive from any peroxides. (Note: the ether floats).