on-collumn DNASe in RNA purification - (Dec/28/2006 )
I am using the promega kit for RNA purification (SV-total system). Now this kit comes with an on-collumn DNAse which seems to reove a lot of DNA (i dont see any on gel), but...i STILL get, almost always, an RT-PCR product in my no-reverse-transcribed control. I eleminate this problem by doing another DNASe treatment, after the kit.
so the question: can i improve the on-collumn digestion, say by adding a second dnase step on the collumn? has anyone here tried it? i asked promega and they dont know...and i am not really keen on experimenting myself cause the kit is expensive and i dont want to waste the collumns.
If you are adding a DNASe step after the column purification you're sample should be DNA free so check possible external contamination or incomplete treatment. Always check that the temperature is the correct one inside the tube, this is one of the must common mistake (put another microtube w/water and check the temp of it).
Tnaks for reply
when i add a DNAse step after the procedure, my RNA is really clean. my questin is, how do i improve the on-collumn DNA digestion, so that i will not have to add this extra step? it adds extra time and waste of material, since i need to clean the RNA again after the digestion step!
pass it through the column more slowly or pass it through a second time.