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Another sonication trouble - (Jun/29/2009 )

Iím trying to do a Pol II IP using mouse B cell line PD31 cells. Hereís what Iíve done so far:

1. Cross link 10^7 for 10 min using 1% formaldehyde at room temp. Wash and resuspend in 1.5 mL lysis buffer. Sonicate using Bioruptor on high setting 30íí on/off up to one hour and no significant shearing occurs.
2. Same as (1) except crosslink on ice for 10 min. Again, no significant shearing after 1 hour.
3. Same as (1) except crosslink on ice for 5 min. Still no significant sonication after 1 hour. So I thought maybe I have too much cytoplasmic debris inhibiting sonication.
4. Cross link 10^7 cells for 10 min using 1% formaldehyde at room temp. Isolate nuclei. Resuspend in 2 mL lysis buffer. Sonicate for 30 min with 30 sec on/off, big smear from 7 kb to 200 bp.

Iím stumped. I donít know what else to do. Iíve used another Bioruptor with the same results so itís not the Bioruptor.

Please help!!

-SheilaTeves-

SheilaTeves on Jun 29 2009, 02:49 PM said:

Iím trying to do a Pol II IP using mouse B cell line PD31 cells. Hereís what Iíve done so far:

1. Cross link 10^7 for 10 min using 1% formaldehyde at room temp. Wash and resuspend in 1.5 mL lysis buffer. Sonicate using Bioruptor on high setting 30íí on/off up to one hour and no significant shearing occurs.
2. Same as (1) except crosslink on ice for 10 min. Again, no significant shearing after 1 hour.
3. Same as (1) except crosslink on ice for 5 min. Still no significant sonication after 1 hour. So I thought maybe I have too much cytoplasmic debris inhibiting sonication.
4. Cross link 10^7 cells for 10 min using 1% formaldehyde at room temp. Isolate nuclei. Resuspend in 2 mL lysis buffer. Sonicate for 30 min with 30 sec on/off, big smear from 7 kb to 200 bp.

Iím stumped. I donít know what else to do. Iíve used another Bioruptor with the same results so itís not the Bioruptor.

Please help!!

1% formaldehyde, 10 minutes, RT should work.

Then..

1. Split your lysates in 300 ul aliquots.
2. Find Diagenode Bioruptor.
3. Do 30 sec on/off for 20-30 minutes.
4. Run the gel.
5. 500bp-2Kb smear will be visible.

-cellcounter-

SheilaTeves on Jun 29 2009, 03:49 PM said:

Iím trying to do a Pol II IP using mouse B cell line PD31 cells. Hereís what Iíve done so far:

1. Cross link 10^7 for 10 min using 1% formaldehyde at room temp. Wash and resuspend in 1.5 mL lysis buffer. Sonicate using Bioruptor on high setting 30íí on/off up to one hour and no significant shearing occurs.
2. Same as (1) except crosslink on ice for 10 min. Again, no significant shearing after 1 hour.
3. Same as (1) except crosslink on ice for 5 min. Still no significant sonication after 1 hour. So I thought maybe I have too much cytoplasmic debris inhibiting sonication.
4. Cross link 10^7 cells for 10 min using 1% formaldehyde at room temp. Isolate nuclei. Resuspend in 2 mL lysis buffer. Sonicate for 30 min with 30 sec on/off, big smear from 7 kb to 200 bp.

Iím stumped. I donít know what else to do. Iíve used another Bioruptor with the same results so itís not the Bioruptor.

Please help!!


I would agree with cell counter. Split your lysate into smaller aliquots. The bioruptor definitely works better with smaller volumes. I typically shear in 100ul (with cells from 1 well of a 6-well plate;5 X 10^5 or so).

-KPDE-