problem of DNA sonication in CHIP assay - (Dec/20/2006 )
I'm struggling on the CHIP assay now. There is a problem I can’t solve. No matter how hard I tried, I can only sheared the DNA to fragments between 4000 to 200 bp, not the classic 1000 to 200 bp. I have tried to reduce the cell number; to increase the sonication time and power; to change sonicator; change cell type. All of them didn’t work.
The following is the protocol I finally used to shear DNA.
1. grow Hela cell to 1 million in 10 cm dish for one sonication
2. 1% formaldehyde crosslink for 10 min at RT, and 125 mM Glycine stop crosslink for 5min at RT
3. lyse cells in 3 mL CHIP lysis buffer (20 mM Tris-HCl pH8.0, 75 mM NaCl, 75 mM KCl, 1 mM EDTA pH8.0, 1 mM EGTA, 1% Triton X-100, 10% glycerol, 1 mM DTT, Protease Inhibitors)
4. collect the cell pellet after lysis by centrifuge, and shear the DNA in 3 ml the same buffer CHIP lysis buffer. 15W 30 sec × 10 times
5. reverse link by incubate the 20 µl sonicated cell lysate with 200μl Elution buffer (1% SDS, 100 mM NaHCO3) and 12 μl 5M NaCl at 65ºC for 4 hr
1. if I sheared the DNA without crosslinking, the DNA was very easy to be sheared to short fragments.
2. if I increased the sonicator’s power higher than 15W, there would be a lot of bubble and heat produced
Can anyone help me figure out what is wrong with my CHIP assay? Can DNA really be sheared to 1000~200bp with crosslinking? Thanks very much.
Yes, you can.
Here's some suggestions:
0. Prep your samples on ICE.
1. Xlink with RT Formaldehyde for less time, say 5 min.
2. Add COLD glycine for 10m.
3. I use a lysis buffer with PMSF, Sodium Butyrate (HDAC inhibitor), and MPI.
4. Make sure your sonicator probe is working properly (we;ve found that they can sometime foul up and need adjusting).
6. compate your protocol with the one at Cold Spring Harbor Protocols (CSH ChiP Protocol)
Thanks for your nice suggestion very much. I'm going to try it.