3 replies to this topic

[bowmanta]

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)?

[Clare]

bowmanta, on 10 September 2010 - 04:18 AM, 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

[rkay447]

Clare, on 10 September 2010 - 05:54 AM, said:

bowmanta, on 10 September 2010 - 04:18 AM, 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.

[Rupam]

The precipitate you found is glycogen definately and you have to add more salt for clear isolation.

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