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Sorry! I am doing ChIP experiment from liver tissue, and so I am interested in collecting all DNA that is bound to my protein of interest. After the immunoprecipitation, the elution buffer that I am using to elute the antibody+ protein + chromatin from the magnetic beads is 50mM Tris-Hcl pH=8, 10mM EDTA and 1% SDS, O/N, at 65C. But I have seen many protocols where they use 1% SDS and 0´1M NaHCO3 for 30 minutes, they discard the beads and they continue with the reverse crosslinking, just with the eluate. As in my case I didn´t get enough material, I was thinking that maybe my elution buffer is not very strong and maybe I am loosing some DNA bound to beads. And for the DNA purification on the Qiagen kit I elute the samples with mQ water.Thank you very much!
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In Topic: Low DNA concentration for sequencing
13 June 2013 - 01:30 AM
Hi, I realize I am mixing many things in my questions, so it´s not easy to follow. Sorry! I am doing ChIP experiment from liver tissue, and so I am interested in collecting all DNA that is bound to my protein of interest. After the immunoprecipitation, the elution buffer that I am using to elute the antibody+ protein + chromatin from the magnetic beads is 50mM Tris-Hcl pH=8, 10mM EDTA and 1% SDS, O/N, at 65C. But I have seen many protocols where they use 1% SDS and 0´1M NaHCO3 for 30 minutes, they discard the beads and they continue with the reverse crosslinking, just with the eluate. As in my case I didn´t get enough material, I was thinking that maybe my elution buffer is not very strong and maybe I am loosing some DNA bound to beads. And for the DNA purification on the Qiagen kit I elute the samples with mQ water.Thank you very much!
Sorry! I am doing ChIP experiment from liver tissue, and so I am interested in collecting all DNA that is bound to my protein of interest. After the immunoprecipitation, the elution buffer that I am using to elute the antibody+ protein + chromatin from the magnetic beads is 50mM Tris-Hcl pH=8, 10mM EDTA and 1% SDS, O/N, at 65C. But I have seen many protocols where they use 1% SDS and 0´1M NaHCO3 for 30 minutes, they discard the beads and they continue with the reverse crosslinking, just with the eluate. As in my case I didn´t get enough material, I was thinking that maybe my elution buffer is not very strong and maybe I am loosing some DNA bound to beads. And for the DNA purification on the Qiagen kit I elute the samples with mQ water.Thank you very much!
In Topic: How many ug of DNA per ChIP is recommendable.
22 October 2012 - 07:15 AM
Great, thanks a lot. 
In Topic: How many ug of DNA per ChIP is recommendable.
18 October 2012 - 07:40 AM
Sorry, I have another question. How do you quantify the DNA? I reversed the crosslinking of a small aliquot of DNA, and measured the concentration before and after purification with the Qiagen kit, and found a big difference in the concentration. I guess the value I get from the purified DNA might be more accurate, without the proteins and the buffer that can interfere. From a tissue sample of 50mg (liver) is it ok if I get like 60ug of DNA? thanks!!
In Topic: How many ug of DNA per ChIP is recommendable.
14 October 2012 - 11:48 PM
I am trying to ChIP some coregulators that don´t attach directly to DNA, so I guess I should use maybe more material that I was using. I am new in this and didn´t have any clue about how much material to use per IP! now I know and can start testing! thanks a lot!
now I know and can start testing! thanks a lot!
