I use Sonics VibraCell VCX 130 to shear chromatin. I did it by 60% amplitude, 15 s on, 50 s off, totally 24 cycles. I always get from 75 to 300 bp fragment, mainly around 150 bp. How can I get larger fragments? I used tissue as starting material. And I use buffers LB1, LB2, LB3 from Young's protocol. And I also tried the IP buffer from fast ChIP protocol. Also I cannot get larger fragments. What's wrong? Can anyone help me?
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#2
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Posted 19 August 2009 - 05:23 PM
rqliang, on Aug 20 2009, 07:51 AM, said:
I use Sonics VibraCell VCX 130 to shear chromatin. I did it by 60% amplitude, 15 s on, 50 s off, totally 24 cycles. I always get from 75 to 300 bp fragment, mainly around 150 bp. How can I get larger fragments? I used tissue as starting material. And I use buffers LB1, LB2, LB3 from Young's protocol. And I also tried the IP buffer from fast ChIP protocol. Also I cannot get larger fragments. What's wrong? Can anyone help me?
Reduce the number of cycles? Reduce the amplitude?
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#3
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Posted 19 August 2009 - 09:42 PM
With a peak around 150bp, my guess is your looking at RNA. Try treating your samples with RNase and see if what you're looking at goes away. The DNA may be a fainter smear and you're not seeing it with the bright RNA smear.
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Posted 20 August 2009 - 07:43 AM
I don't think so. I treated the chromatin with RNase A and proteinase K before I ran the gel. And I did qPCR check using that sonicated sample. It's okay. But I need 500 bp fragments.
KPDE, on Aug 19 2009, 10:42 PM, said:
With a peak around 150bp, my guess is your looking at RNA. Try treating your samples with RNase and see if what you're looking at goes away. The DNA may be a fainter smear and you're not seeing it with the bright RNA smear.
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Posted 20 August 2009 - 07:46 AM
I tried to reduce the number of cycles. I got two smear. One is around 100 bp-300 bp. The orther is more than 2 kb. Reduce the amplitude I got the same thing.
swanny, on Aug 19 2009, 06:23 PM, said:
Reduce the number of cycles? Reduce the amplitude?
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Posted 19 October 2009 - 10:48 AM
rqliang, on Aug 20 2009, 11:46 AM, said:
I tried to reduce the number of cycles. I got two smear. One is around 100 bp-300 bp. The orther is more than 2 kb. Reduce the amplitude I got the same thing.
swanny, on Aug 19 2009, 06:23 PM, said:
Reduce the number of cycles? Reduce the amplitude?
rqliang -
When you're shearing DNA for IPs, the Volume of the DNA solution is as important as the time of shearing or the intensity of the sonicator. Also interesting is that the concentration of DNA in the solution is not very important at all. So he smaller the volume, the more control over exact size you will have. I learned this after many frustrating changes in my sonication results. Now I very carefully measure the volume of the sheared DNA solution into 1 ml aliquots and sonicate each separately. (BTW, for me 4x20 seconds at 20% intensity is the perfect amount, but I use a different sonicator).
Good luck.
