Hi,
Any help on this would be greatly appreciated!
I use the NEB mastermix library prep kit to make libraries from my ChIP samples. For the past month or so I have had poor yield for my ChIP libraries (but not for the input libraries). One thing I've noticed is that during the ligation step when I add the ligase buffer to ChIP samples I get a white precipitate which turns the sample cloudy. On the other hand when I add the ligase buffer to the input samples the sample stays clear. Any ideas why the precipitate forms and if it could affect the efficiency of adaptor ligation?
I tried asking technical support at NEB and they suggest the following:
'It sounds like maybe there is still protein left in the sample, maybe the reverse crosslinking did not get completed? It sounds like the protein in the sample is most likely interfering with the Blunt/TA ligase formulation. I would suggest to check your sample as this may interfere with the ligation and PCR step.'
I reverse crosslink for 5 hours with NaCl at 65C and treat with Proteinase K overnight followed by phenol-chloroform extraction and ethanol precipitation. I treat my input and ChIP samples the same so not sure why protein contamination would not also happen in my input sample.
Any help on this would be greatly appreciated!
I use the NEB mastermix library prep kit to make libraries from my ChIP samples. For the past month or so I have had poor yield for my ChIP libraries (but not for the input libraries). One thing I've noticed is that during the ligation step when I add the ligase buffer to ChIP samples I get a white precipitate which turns the sample cloudy. On the other hand when I add the ligase buffer to the input samples the sample stays clear. Any ideas why the precipitate forms and if it could affect the efficiency of adaptor ligation?
I tried asking technical support at NEB and they suggest the following:
'It sounds like maybe there is still protein left in the sample, maybe the reverse crosslinking did not get completed? It sounds like the protein in the sample is most likely interfering with the Blunt/TA ligase formulation. I would suggest to check your sample as this may interfere with the ligation and PCR step.'
I reverse crosslink for 5 hours with NaCl at 65C and treat with Proteinase K overnight followed by phenol-chloroform extraction and ethanol precipitation. I treat my input and ChIP samples the same so not sure why protein contamination would not also happen in my input sample.
Comment