Inconsistent ChIP PCR - PCR bands vary from same DNA sample (Oct/14/2008 )
I am doing ChIP comparing Wt and KO. Briefly, I collect my samples, crosslink for 10", wash, lyse cells and nuclei, sonicate (I get a smear of upto 1500kb), perform my IP using an antibody against methlyated Histone H3 and purify the DNA with Sigma PCR purification kit. My problem is that while the first couple of times the ChIPped DNA from the KO showed no bands (which is what I expected), the next times the PCR with the same DNA gives bands almost equal or just a little less to the Wt....how can amplification be different from the same DNA sample????????????
My PCR conditions:
38 cycles, 58 degree Annealing temperature, reaction volume of 50 microliter
A few additional notes which I wonder if is the reason behind this:
1.The DNA is very dilute....I could not quantify it by spectrophotometer. Could this result in inconsistent results?I am now thinking of using Trizol and Phase-lock gel system to increase the DNA purified.
2. I had previously stored the DNA at -20. Since I wondered if repeated freeze thaw could make confirmational changes and thus lead to variations in PCR, I now store the purified DNA at 4 degrees...What is the optimal temp to store genomic DNA?
3.Is it a primer problem?My primers have been published for ChIP in atleast 2 papers. I get single band at the expected band size.No non-specific bands, although something which look like primer dimers(below 100bp) are visible.
I would be very appreciative of any answers. I have no clue how to proceed. I am seriously running short of antibodies (now out of stock) and time(finishing my PhD), so I can not repeat the experiment too many times.
Inconsistence among repeated PCRs is quite common especially if you are repeating the same PCR time and time again and using high cycle number. It is very easy to get cross contamination. Using aerosol resistant tips helps a lot. Have you included water controls?
Could you explain a little bit more about the inconsistence among repeated PCRs? I had this problem as well. Sometimes I also saw stronger band amplified from my negative control than the IPed sample. When you talk about high cycle numer, how many cycles will you consider as a high cycle number? Do you have any suggestions about the ChIP PCR primer design, PCR reaction design?
I don't think I contaminated my samples when I repeated the same PCRs. However, the inconsistent results are just so frustrating...
Thank you very much.
I have the same problem....when I first do the pcr I have good results but when I try to repeat it, I have bands almost everywhere, including the pcr negative control.
I believe that it is amplicon contamination, although I use filter tips. Especially, for a pcr product that is very small (about 60 bp), I have inconsistent results almost every time.