salt for DNA precipitation - DNA purification (Sep/10/2010 )
I just joined a new lab and was told to follow the protocol as it is written. While I am a nervous-nelly anyway, I did as asked. The problem is that when I started to isolate DNA that I EtOH precipitated at -20C the night before, I realized that I never added salt! I am preparing ChIP DNA and I reversed the crosslinks in the presence of 5M NaCl (0.2M final). I proteinase K digested, PCI extracted then precipitated O/N with 2.5x EtOH and 20ug glycogen. I did see a pellet after cfg 20' 4C but am wondering if this is only glycogen. Did I need to add more salt (NaCl or Na Acetate)?
bowmanta on Fri Sep 10 12:18:55 2010 said:
I just joined a new lab and was told to follow the protocol as it is written. While I am a nervous-nelly anyway, I did as asked. The problem is that when I started to isolate DNA that I EtOH precipitated at -20C the night before, I realized that I never added salt! I am preparing ChIP DNA and I reversed the crosslinks in the presence of 5M NaCl (0.2M final). I proteinase K digested, PCI extracted then precipitated O/N with 2.5x EtOH and 20ug glycogen. I did see a pellet after cfg 20' 4C but am wondering if this is only glycogen. Did I need to add more salt (NaCl or Na Acetate)?
Ooops
I would just add some salt (3M NaoAc) and chuck it back in the freezer. The pellet you saw prob. was glycogen.
Clare
Clare on Fri Sep 10 13:54:58 2010 said:
bowmanta on Fri Sep 10 12:18:55 2010 said:
I just joined a new lab and was told to follow the protocol as it is written. While I am a nervous-nelly anyway, I did as asked. The problem is that when I started to isolate DNA that I EtOH precipitated at -20C the night before, I realized that I never added salt! I am preparing ChIP DNA and I reversed the crosslinks in the presence of 5M NaCl (0.2M final). I proteinase K digested, PCI extracted then precipitated O/N with 2.5x EtOH and 20ug glycogen. I did see a pellet after cfg 20' 4C but am wondering if this is only glycogen. Did I need to add more salt (NaCl or Na Acetate)?
Ooops
I would just add some salt (3M NaoAc) and chuck it back in the freezer. The pellet you saw prob. was glycogen.
Clare
I agree!! You really need the salt to precipitate DNA. Just add the salt, mix and re-freeze.
The precipitate you found is glycogen definately and you have to add more salt for clear isolation.
You can ask these kind of queries and your possible doubts by connecting to wiziq.It is a very helpful site and you can even attempt various test over their to increase your knowledge.Multiple choice questions increase knowledge about the question having one word answer so one of them are multiple choice questions in biology it will really help you.
Regards
Rupam