KPDE, on 06 August 2010 - 08:00 PM, said:
Just out of curiosity, when do the proteinase K digestions happen in each of the conditions. I'm just curious if the digestion happens in the presence of chelex in one condition but not the other.
I wouldn't worry about the 260/280 ratio. Chelex doesn't actually remove anything from your DNA samples other than multivalent cations like Ca2+ and Mg2+ (that, and the raising of the pH above 10, is the main function of chelex in the assay). There should still be a lot of short peptides and amino acids left from the prot k digestion which will throw off your 260/280.
Finally, the EtOH extraction isn't necessary. We put it in the protocol because we were worried about some of the contaminants (protease and phosphatase inhibitors, etc.) causing problems with PCR, since there is no DNA cleanup step in the protocol. I have gotten fairly reliable dilution curves (even including a 1X point) in PCR without doing the EtOH cleanup on input samples.
Thanks Joel! It's great to have the very original author for advice, wow~
I guess my description wasn't very accurate. In fact, I did the chelex extraction on both the non-decorsslinked and decrosslinked samples. After running a gel with those, I directly did prot. K treatment to both of them and run another gel. I think maybe the prot. K digest the high MW bands (possibly DNA with protein??) so they disappeared. But what's troubling to me is that the one only with chelex runs higher than the one with decrosslink and chelex (they both smeared from top to bottom of the gel before prot.K treatment). I don't know how that happened
In addition, my 260/280 raiots were kind of reasonable, around 1.7-1.8 but my 260/230 ratios were horrible with a really high peak at 230. I am concerned if that contributes to any of the results that I have on the gel and would interfere with later PCRs...
And thanks for the advice on the EtOH clean-up, that's really good to know. I was actually thinking if I could use a 20% Chelex and add equal volume to extract so i can have concentrated samples for loading on gel for optimizing conditions. But I'm not sure if the chelex concentration is really crucial? Any suggestion on this?
I really appreciate any advice that you will offer
Edited by Shan, 07 August 2010 - 12:38 PM.