20 replies to this topic

[timjim]

Hi guys,

I was in the forum long time ago and then there was a crisis and now I became a new member. =) Anyway, I have some difficulties shearing my DNA and I cant seem to get the size I want (200 to 500 bp). I am using Bioruptor and the settings are max for 20 cycles (30 sec sonication, 30 sec ice) - means it is 20 minutes.

My cell number is roughly 2.5 x 10^6. But I still cant get the sheared DNA.

Should I really use MNase treatment? or should I reduce my cell number? or should I increase my SDS lysis buffer?

Thanks alot for any input.

cheers.

[pcrman]

Welcome back Timjim!

I've never used a bioruptor and could not offer much help. The sonication time required by bioruptor is unusually longer than probe based sonicators. I read the bioruptor protocol and it seems you follow it quite well. The only thing you can do probably is to further increase the cycle. Some cells are just more refractory to sonication.

[Clare]

Hello

We use a Bioruptor too. I am a little concerned that you aren't seeing sheared DNA after 20 minutes!! We cross-link our cells in formaldehyde at a final conc. of 1% for 10 mins. Then we sonicate at approx. 1 million cells per 30ul (as recommended by the company). For us, approx. 7 minutes is fine

What are your cross-linking conditions?

Clare

timjim, on Jun 10 2009, 10:24 PM, said:

Hi guys,

I was in the forum long time ago and then there was a crisis and now I became a new member. =) Anyway, I have some difficulties shearing my DNA and I cant seem to get the size I want (200 to 500 bp). I am using Bioruptor and the settings are max for 20 cycles (30 sec sonication, 30 sec ice) - means it is 20 minutes.

My cell number is roughly 2.5 x 10^6. But I still cant get the sheared DNA.

Should I really use MNase treatment? or should I reduce my cell number? or should I increase my SDS lysis buffer?

Thanks alot for any input.

cheers.

[timjim]

Hi Clare,

Thanks for your input. I am using the same fixation as well for 1% formaldehyde and then glycine to stop the fixation. But I have to admit, probably it is longer than 10 minutes. Maybe 15 or 20 minutes and I read the bioruptor manual and it says that the longer the fixation, the longer is the sonication. So maybe the fixation is the problem here.

pcrman,

Thanks for the welcoming back. I am back finally again.

What is the maximum cycle for sonication? I dont want my DNA to be less than 100bp!

thanks alot guys

[Clare]

Hi again

You're right - increased fixation = increased sonication. Are you doing TF or histone ChIPs?
Clare

timjim, on Jun 11 2009, 10:21 AM, said:

Hi Clare,

Thanks for your input. I am using the same fixation as well for 1% formaldehyde and then glycine to stop the fixation. But I have to admit, probably it is longer than 10 minutes. Maybe 15 or 20 minutes and I read the bioruptor manual and it says that the longer the fixation, the longer is the sonication. So maybe the fixation is the problem here.

pcrman,

Thanks for the welcoming back. I am back finally again.

What is the maximum cycle for sonication? I dont want my DNA to be less than 100bp!

thanks alot guys

[timjim]

Hi Clare,

i am doing Histone ChIP. What are the tips for this unique technique? Thanks!

[Clare]

Easy then There's no way to need to fix for 15-20 mins for histones! What cells are you using? I suggest you fix for 10 mins only, then sonicate for 7 mins (high, 30 sec on/off). We use human T cells, neutrophils and myeloid blast cells for our ChIPs and 7 mins is fine. You may want to try 5 mins, 7 and 10 or something. And try the 1 mill cells/30ul and see how you go

Let us know how you get on!

Clare

timjim, on Jun 11 2009, 10:53 AM, said:

Hi Clare,

i am doing Histone ChIP. What are the tips for this unique technique? Thanks!

[KPDE]

timjim, on Jun 11 2009, 02:21 AM, said:

Hi Clare,

Thanks for your input. I am using the same fixation as well for 1% formaldehyde and then glycine to stop the fixation. But I have to admit, probably it is longer than 10 minutes. Maybe 15 or 20 minutes and I read the bioruptor manual and it says that the longer the fixation, the longer is the sonication. So maybe the fixation is the problem here.

pcrman,

Thanks for the welcoming back. I am back finally again.

What is the maximum cycle for sonication? I dont want my DNA to be less than 100bp!

thanks alot guys

Do you use thin walled tubes (like the 0.5ml tubes you would use for PCR) for sonication?  We switched to those and the efficiency of sonication increased dramatically.  Also, we rigged up a cooling apparatus (a parastaltic pump rigged to copper coil in a bucket of ice water with tubes circulating the water through the bioruptor; it's actually really simple to set up) so that we don't have to use ice in the bioruptor.  The ice was severely decreasing our sonication efficiency.

[timjim]

Hi guys, here is my latest shearing.

1 = roughly 1.8 x 10^6
2 = dilution of 2
4 = dilution of 4

20 min is 20 cycles of shearing and likewise 30 min is 30 cycles

I am quite skeptical with the band at the bottom. Are those too much shearing effect? And the shadow effect is due to the loading dye.

What do you guys think? Can I proceed? But I dont like the smearing above 1.5kb.
Thanks!

Attached Thumbnails

• 

Edited by timjim, 12 June 2009 - 01:20 AM.

[KPDE]

timjim, on Jun 12 2009, 02:19 AM, said:

Hi guys, here is my latest shearing.

1 = roughly 1.8 x 10^6
2 = dilution of 2
4 = dilution of 4

20 min is 20 cycles of shearing and likewise 30 min is 30 cycles

I am quite skeptical with the band at the bottom. Are those too much shearing effect? And the shadow effect is due to the loading dye.

What do you guys think? Can I proceed? But I dont like the smearing above 1.5kb.
Thanks!

How high above 1.5kb does the smearing go?  If it only goes as far as 2kb or so I would proceed.  You may not be able to get much better than that.  As far as the band at the bottom, I highly doubt that it is due to overshearing as I get that band regardless of how much shearing I do.  It's probably RNA fragments or something like that since it is at such a low MW.

[timjim]

thanks for the input! But it is really hard just to shear the cells! I am still working on it.

But someone in the lab did 40 minutes (40 cycles) of sonication using Bioruptor and I can see a strong band at around 200 to 500 bp. No smearing at all! It is clean shearing for me. Only 200 to 500 bp. But I do think 40 minutes are abit too long? Especially one is using Bioruptor? We always do 10 minutes interval and change the ice. I wonder if that makes the shearing efficiency to drop?

Thanks!

[KPDE]

timjim, on Jun 23 2009, 04:21 AM, said:

thanks for the input! But it is really hard just to shear the cells! I am still working on it.

But someone in the lab did 40 minutes (40 cycles) of sonication using Bioruptor and I can see a strong band at around 200 to 500 bp. No smearing at all! It is clean shearing for me. Only 200 to 500 bp. But I do think 40 minutes are abit too long? Especially one is using Bioruptor? We always do 10 minutes interval and change the ice. I wonder if that makes the shearing efficiency to drop?

Thanks!

If someone did 40 minutes without changing the ice, I'm guessing the ice was melted by the end.  What probably allowed the shearing to work was that all the ice melted.  The ice severely reduces sonication efficiency for us.  In fact we found it impossible to get good shearing with ice in the bath.  You might try sonication with short intervals, replacing the water with ice cold water each time.  As I mentioned above, we developed a simple water cooling system and that solved all our shearing problems.

[timjim]

well, we change the ice every 10 minutes (so in total it is 40 mins), sometimes we get sheared band sometimes we dont. And they are the same samples!!! I really dont understand the cells sometimes. Getting irritated by the stupid cells. =p

I dont really understand the cooling system, so basically, one should just pump the water out and replace with cold water? I would like to make such cooling system too. Well, you can even buy it from Diagenode....

[hamidk]

Hi Timjim,

The issue with using a sonicator for chromatin shearing is with reproducibility of settings with different cell lines. All shearing conditions have to be optimized for each cell line which can be quite a laborious task in itself. Thermal damage is a major issue when dealing with smaller shearing sizes. Thermal denaturation of the the proteins, non-specific and biased shearing, thermal acetlyation issues are commonly noticed.
Have you heard of the Covaris AFA technology for non-contact, isothermal chromatin shearing? Here are some protocols using the Covaris technology http://covarisinc.co...a-shearing.html. Let me know if you need further details.  Good luck.

hamid

[Radish]

hamidk, on Jul 12 2009, 05:42 PM, said:

Hi Timjim,

The issue with using a sonicator for chromatin shearing is with reproducibility of settings with different cell lines. All shearing conditions have to be optimized for each cell line which can be quite a laborious task in itself. Thermal damage is a major issue when dealing with smaller shearing sizes. Thermal denaturation of the the proteins, non-specific and biased shearing, thermal acetlyation issues are commonly noticed.
Have you heard of the Covaris AFA technology for non-contact, isothermal chromatin shearing? Here are some protocols using the Covaris technology http://covarisinc.co...a-shearing.html. Let me know if you need further details.  Good luck.

hamid

Hello to you all


I have a dumb question, in terms of sonication efficiency what is the difference between 30 sec on/30 sec off cycles and smaller cycles, like 15 sec on/15 sec off?

Thanks