ChIP PCR help, maybe is the problem of SDS. - (Jul/14/2007 )
I have just begun to do ChIP.
After ChIP, I use the Qiagen Quick PCR purification kit to purify DNA. Using the purified DNA to do PCR for 40 cycles, I met a very strange problem: The same sample can not get the same result. I mean this time I can get very strong band, but using the same sample (purified DNA) I can not get a little band next time. But using the input DNA, I can get the good and consistent result.
Is the problem of SDS, but I have purified the elution sample. I have tried many times, I was almost crazy.
Is anybody meeting this problem? Please help me.
Below is the buffer I used:
1. Lysis buffer:
1% SDS
50mM Tris pH 8.0
10mM EDTA
2. Elution buffer:
25mM Tris-Cl pH7.5
10mM EDTA
0.5% SDS
Is the elution buffer you gave the final buffer you used to elute DNA from Qiagen purification kit? If yes, why not use the EB buffer which is provided with the kit and does not contain SDS?
Thank you much!
I added the elution buffer to the last step of washing, vortexed, and incubated at 65 degree overnight. Then heated to 95oC for 30mins, centrifuged, and the supernatant was purified using Qiagen purification kit. I have tried the EB buffer and water to elute DNA from Qiagen column, but the problem is still there.
There might be problems with either the ChIP step or the Qiaquick purification step. For the Qiaquick purification, I always add 5 ul of 3M NaOAc (pH 5.2) to make sure that the ChIP DNA is bound to the spin column. The Qiagen columns require pH around 7-8 for optimal binding. It may not be a problem for the input DNA because there's much more of it. I use either water or EB with the kit for eluting the DNA.
For the ChIP part, people usually use SDS and NaHCO3 to elute the bound protein-DNA complex and reverse the X-link at 65 degree over night. I've never seen people using a 95 degree step. You might need to pick another gene/region besides your gene of interest, as a internal reference. That will help you figure out if the problem is with the ChIP itself or with the Qiaquick purification.
Good luck.
For the ChIP part, people usually use SDS and NaHCO3 to elute the bound protein-DNA complex and reverse the X-link at 65 degree over night. I've never seen people using a 95 degree step. You might need to pick another gene/region besides your gene of interest, as a internal reference. That will help you figure out if the problem is with the ChIP itself or with the Qiaquick purification.
Good luck.
In fact, elution and reversal of crosslinking can be done by boiling (or heating to 95C) the protein A or G beads with 10% chelex (in H2O) for 10 min. See Nucleic Acids Research 34(1): e2.