I really hope somebody can help me here, I have been trying this for the last 5/6 weeks and getting nowhere..I have a review coming up soon and I really want to have something to show!!
-I have been trying to prepare chromatin by sonication using the Diagenode Standard Bioruptor.
- This chromatin preparation is supposed to be for a ChIP-seq.
-The lysis buffer I have been using is - 50mM Tris-HCL, 150mM NaCl, 5mM EDTA, 0.5% NP-40, 0.5% TritonX100 and PIC.
-Cells cross linked on plate using 16% paraformaldehyde (at 1%) for 15mins at RT and then harvested by scraping.
The way I prepare my samples is as follows:
- Wash/re-suspend 10^7 cells in 1ml of lysis buffer and pipette up and down gently
- Spin at 12,000g for 1mim/4C
- Discard supernatant and re-suspend pellet for a wash - (spin 12,000g/1min/4C)
- Re-suspend pellet in 300ul of lysis buffer again.
- Sonicate 30secON/OFF in bioruptor waterbath sonicator for 10,20,25,30 cycles (I have also done 8,12,16,20 cycles).
- After sonication I spin at 12,000g for 10mins and keep the supernatant.
- I take 500,000 cells worth (~15ul) and process as follows
- Dilute 15ul to 100ul and put in thermocycler overnight at 65C to reverse cross link
- Phenol chloroform extract by diluting the sample to 500ul(in lysis buffer) then adding 500ul for PCIA(25:24:1)..vortex/spin-top speed/2mins
- I repeat this extraction 2 more times
- I then supplement with 3M NaCl to 0.3M, add 1ul of glycogen, 1ml of 100% ethanol and -20C overnight
- The next day I wash twice with 70% ethanol (ice cold - add ethanol, light vortex, 5min spin)
- I then let the pellet air dry for 0.5-1hr and resuspend in 30ul of TE
- The sample is then treated with RNaseA - Sometimes I treat with RNaseA during cross link reversal instead.
Things to consider..
- I do not use the TPX tubes that diagenode recommend - Any thoughts on this?? - there doesn't seem to be much difference in the user manual examples.
- My supervisor does not want to use SDS because of a reason I don't really know - Does this effect the IP? - Any thoughts?
- Should I be using SDS?? is it ok as long as the SDS is diluted down when it comes to the IP??
- On average the amount of DNA I get back after PCIA/EtOH is around 3ug in total (I have not assayed the pellet though and I have estimated that in 500,000 HeLa cells there should be ~6.5ug of DNA)
- I have tried Proteinase K treatment also..but I just seem to get a smear the whole way down for all samples.
- Any ideas on assaying the DNA content?..Should I assay the pellet..if so..how?
P.S - Sorry for posting yet another topic on sonication - but the others haven't really helped my situation!!
Edited by j47027, 20 May 2013 - 03:46 PM.