ChIP Assay Problems - Receiving poor data from assay (Oct/22/2010 )

Hello,
I am working incredibly hard to perfect my lab's ChIP assay with their controls prior to moving to the experimental system and hypothesis testing. The problem I have is very low chromatin DNA yields at the end of the experiment, as measured by picogreen (standards for picogreen work). I'm using Rag-/- mouse cells (transgenic line). I've posted buffers below and have attached/linked the specific method. One thing I notice is that during the DNA Elution part, the salt precipitates when placed in the 300 ul Elution buffer that includes the IPs. I thought this might be one problematic area leading to low chromatin DNA yields at the end. Any help is much appreciated!!!!





SDS lysis Buffer: 50 mM Tris-HCl pH 8, containing 1% SDS and 10 mM EDTA; combine 41.5 ml water, 2.5 ml 1M Tris 8, 1 ml 0.5 M EDTA, 5 ml 10% SDS.

ChIP dilution Buffer: 16.7 mM Tris-HCl pH8, containing 167 mM NaCl, 1.2 mM EDTA, 1.1% TX-100; for 20 ml, combine 18.75 ml water, 334 µl 1M Tris 8, 668 µl 5M NaCl, 48 µl 0.5 M EDTA, 220 µl TX-100. Alternative, with NP-40, for 50 ml- 46.8 ml H2O, 835 ul 1M Tris-HCl pH8, 1.67 ml NaCl, 120 ul EDTA, 1.1 ml NP40.

ChIP wash 1: 20 mM Tris-HCl pH 8, containing 150 mM NaCl, 2 mM EDTA, 1% TX-100, 0.1% SDS; combine 46.3 ml water, 1 ml 1M Tris 8, 1.5 ml 5M NaCl, 200 µl 0.5M EDTA, 500 µl TX100, 500 µl 10% SDS.

ChIP wash 2: 20 mM Tris-HCl pH 8, containing 500 mM NaCl, 2 mM EDTA, 1% TX-100, 0.1% SDS; combine 42.8 ml water, 1 ml 1M Tris 8, 5 ml 5M NaCl, 200 µl 0.5M EDTA, 500 µl TX100, 500 µl 10% SDS.

ChIP wash 3: 10 mM Tris-HCl pH 8, containing 0.25 M LiCl, 1 mM EDTA, 1% NP40, 1% sodium deoxycholate; combine 43.5 ml water, 500 µl 1M Tris 8, 5 ml 2.5 M LiCl, 100 µl 0.5 M EDTA, 500 µl NP40, 0.5 g sodium deoxycholate.

ChIP elution buffer: 1% SDS containing 0.1 M NaHCO3; combine 8 ml water, 1 ml 1M NaHCO3, 1 ml 10% SDS.

Are you sure that you can measure single stranded DNA concentration with picogreen ? Beacause you have mainly ssDNA after chelex (95degres, 15min).

doudou30 on Fri Oct 22 15:29:51 2010 said:

I don't elute using Chelax. I skip all steps under "Chelax 100" and proceed to the "DNA elution/workup, old methods" section. I usually do the Qiaquick clean-up, but I am currently trying the Phenol-Chloroform approach (but, I don't think this will make a difference).

I use dynabeads from invitrogen (between 30ul to 200ul), there are very good. And I tried different elution buffers (one with NaHCO3), but the best one was with no comparison : 50mM Tris-HCl, pH 8.0, 10mM EDTA, 1% SDS, with incubation 1h at 55 degres and vortex every 15 min. If you want to try...
Good luck !