What i did was to sonicate 200ul of samples (containing 2million cells). After 5rounds of 30s on/30s off (biorupter), i'll aliquot 20ul out. The remaining samples i'll continue to sonicate, and another 5 rounds later i aliquot 20ul again as the 10rd sample and so on. I repeat this until finally i have 100ul left which will be sonicated for another 5 rounds to make a total of 30 rounds.
After that i added RNase A and reverse the cross links, purify the pcr product using purification kit, and load the sample onto gel. The protocol i used was adapted from the abcam protcol, and apparently has been successful with my colleague on another cell type. I've quoted the protcol here,
"The DNA is purified using a PCR purification kit (add 70 μl of Elution Buffer and proceed to Step 3.2a)
3.2.a. Add 2 μl RNase A (0.5 mg/ml). Heat with shaking at 65°C for 4-5 hr (or overnight) to reverse the cross-links. DNA is purified using a PCR purification kit according to the manufacturer’s instructions. The samples can be frozen and stored at -20°C.
Samples are treated with RNase A as high levels of RNA will interfere with DNA purification when using the PCR purification kit. Yields can be severely reduced as the columns become saturated."
And just to clarify, the 30rds sample were eluted in the same amount even though there were 100ul of sample. So since i loaded equal volume for all, there were more 30rds samples and hence it looks much brighter. But still i can't explain why it looks like there is a band in the 200bp region.
And also, why don't i see the change in smear size as the no. of rounds of sonication increase?
Edited by krystle, 16 February 2011 - 12:52 AM.