ChIP PCR question - (Feb/22/2011 )

Just out of curiosity, when you say you get variability in your end point PCR, do you mean when you re-run the same samples over the data comes out different? Or are you talking about comparing data from different ChIPs? If the former, have you tried running your PCRs in duplicate or even triplicate? Not sure I have a great answer for you, but I'm just trying to figure out where the variability might be coming from.

I reverse xlink by adding rnase a n incubating at 65 for 4 hrs. I think it works because once I forgot to add a buffer b4 xlink and the smear size was really much bigger. When I reverse xlink I get smear around 250-1000bp size.

I have never used RNase A as a reversal method but I can imagine how it may work......For my own edification could you explain why you use this method? I resuspend my beads in 10% Chelex resin and add 20ug of ProK for 45min at 57 degrees followed by a 96 degree incubation for 20min for my elution/reversal method. I think you can substitute TE buffer for the Chelex though.

mighty mouse, i meant i re-run the same samples and the results varies. Nope i've not ran it in duplicate or triplicate before.

chabraha, i use this method because i saw the chip protocol from abcam, and it says that if i wish to purify the samples using those purification columns, i need to remove the rna or else the high levels will interfere with dna purification using columns. On the other hand, if i were to purify samples using phenol/chloroform method, then i should stick to the traditional way of adding proteinase K.

Anyways, just to let you guys know i think i found out the reason for the variability, though i can't be sure yet. I increased the denaturing, annealing, and extension time for my cycles and managed to get a proper set of data where all the positive primers gives band and all primers give band when using input sample. Seems like the polymerase efficiency might be the problem. I usually only face such issue when i need to PCR rather large product, so i'm not sure why this time round when getting 150-300bp product i need to go to such extent. But i'll need to repeat the whole pcr again to see if i can get the same result. But at least, for now the positive controls all seems to be in place.

Now another question, based on this new set of data, i ran my ip sample, mock ip using IgG, and mock ip without Ab.
I had 3 types of data. 1) bands seen on input and my sample ip, but not on IgG mock or beads only mock (good). 2) bands seen on all samples, with the 3 ip samples giving equal brightness. 3) bands seen on input, sample ip and beads only control. IgG mock is clean but not beads only control.

so for 2) and 3), i'm thinking it might be primer problem since the other primers don't have such issue, but i'm not sure. The primers are all specific since there were only single bands observed in all case.
any suggestions as to this issue? thanks alot guys!