Hi,
I have done sonication on my formaldehyde-fixed samples, using covaris kit. Then I took a bit of sheared chromatin to test the shearing efficiency. To reverse the cross-link I add RNAseA- Incubate 37c, 30 min, then Proteinase K was added and incubate ON at 65 C.
The next day I purify the DNA by column purification and Check the size on the bioanalyzer which looks like Fig1 (attached).
Then I did a few more cycles sonication on the samples to make sure that I am totally in the safe side and I continued with IP, after IP, I did the reverse cross-link on samples (IP and Input) based on the following protocol:
Then I did the Column purification of DNA, and run the input samples on Bioanalyzer (High sensitivity Kit), but I got different profiles this time! Fig 2 (Attached)
I run the input samples on an agarose gel, and based on the agarose gel, I have 300 bp band and no large band!
So I am pretty sure that my sonication is fine, but what is the large band in Fig2? Is it because of an incomplete reverse cross-link in the step2?
How could I get rid of this large band before proceeding to library preparation and sequencing?
Since I am afraid that it will affect all the future bioanalyzer experiments! :(
Thanks a lot in advance for your kind consideration and help,
Bests
Hi Mahdieh, sorry if I ask, is there a difference in concentration between the input and ip sample you are running on the bioanalyser? I find often when I run my samples in a high DNA sensitivity chip that if the DNA concentration is too high it won't run properly, hence high molecular peak. I suggest you to try either to run the reverse cross linked input in a not high sensitivity DNA chip or to test several dilutions (serial dilution could make it) of your input in the high sesitivity DNA kit. Hope it helps
Hi,
Which Ab have you sued for IP? it looks like a preferential immunoprecipitation of large fragments over small ones, a well-known phenomena for inactive marks. It is not a real problem, you could re shear IP's samples until desirthe ed size in order to nclude them per library prep. Please check Diagenode protocol for this: https://www.diagenode.com/en/protocols/chip-re-shearing-protocol
I hope that will help,
Irina
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