DNA fragmentation by sonication (for ChIP)
|DNA Fragmentation by Sonication.pdf||40.13 KB|
Greg and Vijay- October 2009. For use with the diagenode bioruptor- located in the dark room in HSE 2nd floor.
1. Fill up bioruptor tank with ice.
2. Take samples out of freezer and keep them on dry ice- they must not thaw!
3. Prepare fresh a stock of complete lysis buffer: (N+1)x120ul lysis buffer +NaBu (20ul/ml) + PI (10ul/ml) + PMSF (10ul/ml)- keep at RT.
4. Add 120ul of complete lysis buffer to each sample- allow the pellet to thaw at room temperature into the buffer (1-2 min). Resuspend the pellet until the solution is clear- avoid forming bubbles.
5. Incubate on ice for 5 minutes.
6. Transfer samples to low retention 0.5 ml tubes (try to take everything).
a. Remove ice and fill tank to line with ice cold water.
b. 20 cycles of 30" ON/OFF high power setting
Note- the number of pulses is a function of cell/condition type and needs optimization for your conditions.
c. Keep tank cold by adding a little bit of ice every 4 cycles of sonication. Quick spin samples each time you remove them from the bioruptor to collect the liquid at the bottom of the tube (gives more efficient sonication).
8. Spin samples briefly- keep on ice
Note- SDS will precipitate with low temperature.
9. Take out 2.5ul make a 10-fold dilution in TE (25ul), keep 15ul for quantification.
10. Take 10ul from above- incubate at 98°C for 10 minutes in heat block (quick decrosslinking).
11. Add 2-3ul 6x loading dye and load 6ul onto 1.2% pre-cast agarose gel.
Note- load at the same time the positive control: sonicated sample and DNA ladder.
12. Run gel at 100mV for 15-30 minutes
13. Staining: prepare 50mls TAE with 2.5ul of SYBR gold and soak the gel in the dark for at least 10 minutes on a shaker.
14. Wash the gel in TAE for 2 minutes and read on UV transilluminator.
Note- wash your gloves before touching the gel.
--> The 100% DNA band should stand blow the 1kb with the majority between 200 and 600bp. If this is not the case, do 2-4 more cycles of sonication.