Why my ChIP PCR results are always unrepeatable? even from the same sample. - (Jan/16/2007 )
Hello, everyone, I did ChIP on Transcript factor, Co-IP has confirmed that the IP works. However, after I isolated the DNA and run PCR, it sometimes gave signalling on ChIP sample, but sometimes not. Furthermore, the No-Ab control also had a certain possibility to give out positive signalling.
I have troubleshot for the contamination, but the Mock works well without giving any signalling, it seems there is a kind of unequal distribution of DNA in the sample solution, but there is no reason because I purify the sample from PCR purificaiton Kit, I have worked several monthes to try to figure out what is the point, but still can not get a clear answer.
Let me describe the last several steps I did to purify the DNA, the final elution buffer for the beads wash is TE with 1%SDS, after elution from the Protein A beads, I add Proteinase K and incubate it in 65 degree for over 6 hours and then add RNAse and incubate in 45 for 2 hours, use Qiaquick PCR purification Kit to purify the mixture, then 15ul PCR, the temperature setting is 94-72-58-4, cycles number is around 37-39, then run the gel, the results killed me too many times.
I mix the PCR reagent together and seperate it into different PCR tubes, then I take 2ul sample solution fromt the same tube, that is the only difference, there seems no reason for any difference between two PCR tubes, but usually they are one positive one negative.
Did anyone meet this kind of problem before? Please share your opinions! Thanks!
In addition, I use zebrafish whole embryo to do the ChIP, Now I suspect the uncleaned Yolk material.
try mixing your DNA very well by tapping the tube or even brief vortexing. It could be that the DNA is not evenly distributed. I had similar experience where i ran 3 replicates of the same sample and some gave product some did not.
good luck
Hi, I also met this problem. I tried many times but still not successful. Before seeing your poster, I just wondering if it the problem of proteinase [I have not use the proteinase].
Have you figure out what's the problem of ChIP PCR. could you share me with your experience?
I have troubleshot for the contamination, but the Mock works well without giving any signalling, it seems there is a kind of unequal distribution of DNA in the sample solution, but there is no reason because I purify the sample from PCR purificaiton Kit, I have worked several monthes to try to figure out what is the point, but still can not get a clear answer.
Let me describe the last several steps I did to purify the DNA, the final elution buffer for the beads wash is TE with 1%SDS, after elution from the Protein A beads, I add Proteinase K and incubate it in 65 degree for over 6 hours and then add RNAse and incubate in 45 for 2 hours, use Qiaquick PCR purification Kit to purify the mixture, then 15ul PCR, the temperature setting is 94-72-58-4, cycles number is around 37-39, then run the gel, the results killed me too many times.
I mix the PCR reagent together and seperate it into different PCR tubes, then I take 2ul sample solution fromt the same tube, that is the only difference, there seems no reason for any difference between two PCR tubes, but usually they are one positive one negative.
Did anyone meet this kind of problem before? Please share your opinions! Thanks!
In addition, I use zebrafish whole embryo to do the ChIP, Now I suspect the uncleaned Yolk material.
Do you have the PCR tubes right next to each other on the thermocycler block? Is it possible that your block isn't keeping an even temp from one side to the other?
I have troubleshot for the contamination, but the Mock works well without giving any signalling, it seems there is a kind of unequal distribution of DNA in the sample solution, but there is no reason because I purify the sample from PCR purificaiton Kit, I have worked several monthes to try to figure out what is the point, but still can not get a clear answer.
Let me describe the last several steps I did to purify the DNA, the final elution buffer for the beads wash is TE with 1%SDS, after elution from the Protein A beads, I add Proteinase K and incubate it in 65 degree for over 6 hours and then add RNAse and incubate in 45 for 2 hours, use Qiaquick PCR purification Kit to purify the mixture, then 15ul PCR, the temperature setting is 94-72-58-4, cycles number is around 37-39, then run the gel, the results killed me too many times.
I mix the PCR reagent together and seperate it into different PCR tubes, then I take 2ul sample solution fromt the same tube, that is the only difference, there seems no reason for any difference between two PCR tubes, but usually they are one positive one negative.
Did anyone meet this kind of problem before? Please share your opinions! Thanks!
In addition, I use zebrafish whole embryo to do the ChIP, Now I suspect the uncleaned Yolk material.
I have met the same problem, are you sure the PCR result is specific? I discarded my results and design new primers because I thought the bands in my gel were non-specific.
I have tried using different wells of thermocycler block. It seems not the reason of the PCR thermocycler, because the "input" samples always gave the clear and consistent bands.
Last week I also tried using pronase digestion before purification using Qiagen kit, but the problem is still there. Could you give me some suggestions?
Thanks!
[quote name='KPDE' date='Oct 2 2007, 09:58 AM' post='113164']
Do you have the PCR tubes right next to each other on the thermocycler block? Is it possible that your block isn't keeping an even temp from one side to the other?
Last week I also tried using pronase digestion before purification using Qiagen kit, but the problem is still there. Could you give me some suggestions?
Thanks!
When you get signal for the IP, is it always at about the same level for a given number of cycles? If the amount of signal you get is all over the place could it be that you are getting some kind of contamination.
sounds like contamination to me, possibly excess salts. When you do your final DNA clean up, instead of immeadeately using a Qiaquick column, try a Phenol/Chloroform extractration, or even a direct ethanol precipitation followed by Chelex-100 treatment. Washing multiple times with 70% Ethanol will also help remove excess salts.
Sorry to hear you having trouble. ChIP is not trivial. When your experiments stop working - sometimes throwing out all your reagents and starting over is another strategy. 
Seems to have worked for me recently... lol