ChIP purification method
Posted 27 July 2010 - 06:47 AM
Has anyone had problems using column based purification in their ChIP assay? I have DNA before purification but obviously it shows contamination and then after purification and treatment with RNase the DNA is gone? I think it may be blocking up the columns. Has anyone tried phenol purification for ChIP?
Posted 27 July 2010 - 12:07 PM
Edited by biznatch, 27 July 2010 - 12:11 PM.
Posted 28 July 2010 - 01:31 AM
I am still designing the protocol as no one in my lab has ever done ChIP before. I am still optimising the sonication time but to do this i need to purify it and run it on a gel to check the band size. At the moment after lysis and sonication i am treating the samples with RNase A and proteinase K and heating the sample to 65 degrees C overnight to reverse crosslinks and then purifying using a Qiagen pcr purification kit.
How many cells are you using per ChIP and how long do you sonicate for? It could be that my fragments are still too big and are blocking the column?
Could you possibly send me the protocol?
Posted 28 July 2010 - 07:16 AM
To test sonication, you should be heating (in salt solution, NaCl) overnight first, then treating with RNase then PK. You don't have to purify, just take 20-30uL after PK treatment, add loading buffer and run on a gel.
The PCR purification kit purifies up to 10kb, it's unlikely that your fragments are bigger then that even after a little sonication. When you run the gel do you see anything stuck in the wells?
Edited by biznatch, 28 July 2010 - 07:18 AM.
Posted 28 July 2010 - 07:33 AM
No there is nothing on the gel or stuck in the wells.
Posted 28 July 2010 - 07:50 AM
After proteinase K I've always done phenol/chloroform followed by ethanol precipitation and it's been fine for me.
Posted 28 July 2010 - 08:08 AM
At the end of the ChIP protocol when it comes time to isolate the final DNA for PCR, we do however use columns to purify.
Edited by biznatch, 28 July 2010 - 08:09 AM.