Shearing (sonication) problem for ChIP - Using Bioruptor (Jun/10/2009 )
you should be able to confirm the size via the Tm..........often times primer design programs will give you a theoretical Tm for the PCR product...........you can also calculate this yourself if you do not have a program..........but I think IDT's website can do this for you.
With respect to your question........the possibility exists that you did not expose the gel long enough to see the small products..........and the possibility you bring up is also good...........use a syber green appropriate filter on a chemidoc imager and you'll probably see your products.
i have big problem for preparing a good chromatin for chip analysis.
do you have a good protocol for sonicate activated naive cd4 T cells?
i have in my lab Bioruptor sonicator....i check many lysis buffer and many protocols but i'm not able to have genomic fragment ranging from 500nt to 100nt.
PS: i think my problem is no the lysis (cells are nicely lysated as observed by microscope with trypan blu!!!) but the sonication condition.
I don't use bioruptor, but I usually sonicate my CD4+ lymphocytes at 30% power for 5 cycles, each one of 10seconds. Between each cycle I leave the sample on ice for 30-40sec. In which volume do you sonicate?
are u using RIPA buffer for lysis/sonication?
200ul should be fine, I also do that way. No, I'm using 50ul SDS lysis buffer for lysis (it contains 1% SDS) and then I add 150ul of Chip dilution buffer that contains 0.1% SDS: I obtain fragments around 300-500bp.
I'm sorry, I missed a 0! The chip dilution buffer contains 0.01% SDS. Anyway, you are welcome!
I am standardizing the sonication condition for tissue ChIP assay, I would like to know the fragment (smear +bright band) is from DNA or from RNA contamination, I did RNase treatment; the lane after ladder is the sample without reversing the cross linking. please suggest me.