Protocol for RNase treatment of plasmid for RNA cleanup - (Apr/03/2006 )
I am trying transformation experiements. I have uncut, purified plasmid which looks good on gel, but on nanodrop machine, its purity ratio is not good. I want to make sure that, the extracted DNA is not contaminated with RNA. My plan is to digest my sample with RNAse, and then run on gel. I found RNAase A from Roche in my lab. Can you please give me protocol for above experiment?
thanks very much
i do for midiprep :
resuspend in 500µl TE
Add 1µl RNASE 10mg/ml
Add 2.5µl proteinase K 20mg/ml
add 500µl phenol chlo iAA, vortex and spin 14000rpm 20-30'
pellet the resulting aqueous phase and wash it (etoh step)
resuspend in water or tris 10mM pH 8
Thanks Fred for the protocol but what is chlo iAA?
In fact it comes with the Phenol: it's phenol/chloroform/Isoamyl alcohol in proportion 25/24/1 If I remeber correctly. It is used to get rid of proteins in your solution. If you click on the protocol tab on top of the page you will be able to carry out a serch that will give you plenty of detailed protocols to prepare and purify your DNA with Phenol/chloroform/IAA