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salt for DNA precipitation


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3 replies to this topic

#1 bowmanta

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Posted 10 September 2010 - 04:18 AM

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)?

#2 Clare

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Posted 10 September 2010 - 05:54 AM

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

#3 rkay447

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Posted 10 September 2010 - 09:30 AM


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.

#4 Rupam

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Posted 12 September 2010 - 06:28 AM

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

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