I have methylated my DNA of interest using M. Sss1 methyl transferase from NEB in 20 ul reaction set up as mentioned in their protocol.After this, I tried to purify that DNA by phenol extraction followed by ethanol precipitation. But I did not get any pellet of DNA after this. I started with 4 ug of DNA . What could be the reason? and secondly they have given the protocol for 500 ul reaction set up also and the purification step is mentioned in that protocol only. so, does the small scale methylation (20ul) not require the purification step?
I am attaching the protocol here.
Thanks
I am not sure what the issue is with the protocol, but I think more information is needed. When you say you didn't get a pellet after your precipitation, do you mean that you didn't see anything in the tube or was there nothing when you tried running the final solution on a gel/quantified the amount of DNA? If you have very clean DNA, the pellet may be clear, and hard to see. I think that the issue has less to do with the methylation protocol than your precipitation, since whether or not the protocol has succeeded, you should still recover DNA at the end. I haven't tried methylation of DNA in vitro, but my gut instinct is that the phenol extraction is a step where you may be losing DNA, and I'm not sure that it is fully necessary. Cleaning up most enzymatic reactions does not require a phenol extraction, and either a simple ethanol precipitation or using a PCR clean-up spin column following the heat inactivation will remove the proteins bound to the DNA, as well as removing any unwanted buffers. Perhaps the methylase is very tightly bound to the protein, but I found a paper where the authors chose to skip the phenol extraction sometimes after methylase treatment (see materials and methods section: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3804486/). I think this should be sufficient since the heat inactivation step should denature the DNA and most likely the protein-DNA complex as well if it is still bound at the end of the reaction, so that the ethanol precipitation will give you a clean enough product. Hope that helps!
I am not sure what the issue is with the protocol, but I think more information is needed. When you say you didn't get a pellet after your precipitation, do you mean that you didn't see anything in the tube or was there nothing when you tried running the final solution on a gel/quantified the amount of DNA? If you have very clean DNA, the pellet may be clear, and hard to see. I think that the issue has less to do with the methylation protocol than your precipitation, since whether or not the protocol has succeeded, you should still recover DNA at the end. I haven't tried methylation of DNA in vitro, but my gut instinct is that the phenol extraction is a step where you may be losing DNA, and I'm not sure that it is fully necessary. Cleaning up most enzymatic reactions does not require a phenol extraction, and either a simple ethanol precipitation or using a PCR clean-up spin column following the heat inactivation will remove the proteins bound to the DNA, as well as removing any unwanted buffers. Perhaps the methylase is very tightly bound to the protein, but I found a paper where the authors chose to skip the phenol extraction sometimes after methylase treatment (see materials and methods section: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3804486/). I think this should be sufficient since the heat inactivation step should denature the DNA and most likely the protein-DNA complex as well if it is still bound at the end of the reaction, so that the ethanol precipitation will give you a clean enough product. Hope that helps!
Thank you John. I was unable to see the DNA pellet after phenol extraction .As the initial DNA amount was v.less (2ug) and after phenol extraction some loss of DNA occurs .That is why I did not get any pellet after phenol extraction.. I am convinced with your answer that heat inactivation followed by ethanol precipitation will do. Thanks a lot
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