2 replies to this topic

[SheilaTeves]

I’m trying to do a Pol II IP using mouse B cell line PD31 cells. Here’s what I’ve 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 30’’ 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.

I’m stumped. I don’t know what else to do. I’ve used another Bioruptor with the same results so it’s not the Bioruptor.

Please help!!

[cellcounter]

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

I’m trying to do a Pol II IP using mouse B cell line PD31 cells. Here’s what I’ve 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 30’’ 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.

I’m stumped. I don’t know what else to do. I’ve used another Bioruptor with the same results so it’s 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.

[KPDE]

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

I’m trying to do a Pol II IP using mouse B cell line PD31 cells. Here’s what I’ve 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 30’’ 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.

I’m stumped. I don’t know what else to do. I’ve used another Bioruptor with the same results so it’s 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).