Hi,
I have a question: After a Phenol/Chloroform Extraction I added too much salt: to a mix of 400ul I added 140 ul of 5M NaCl, instead of only 40 ul (1/10) as it was written in the protocol. The DNA pellet fell out immediately. After 20 min I realized my mistake and added an according amount of buffer and ethanol to match the mentioned 1/10th. I am afraid now that the DNA might have been harmed (broken etc.) by the high salt concentration at the beginning. The vector I am using is pretty big (17 kb).
Do you think I cann still use the DNA or should I toss it away?
Thanks,
Lisa
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5 replies to this topic
#2
Adrian K
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Posted 20 January 2011 - 07:26 PM
I think is ok to use... what is your application?
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..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...
"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong
"It's all just DNA. Do it."---phage434
..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...
"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong
"It's all just DNA. Do it."---phage434
#3
Ameya P
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Rervm Cognoscere Cavsas
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Posted 20 January 2011 - 09:30 PM
Lisa86, on 20 January 2011 - 07:04 PM, said:
Hi,
I have a question: After a Phenol/Chloroform Extraction I added too much salt: to a mix of 400ul I added 140 ul of 5M NaCl, instead of only 40 ul (1/10) as it was written in the protocol. The DNA pellet fell out immediately. After 20 min I realized my mistake and added an according amount of buffer and ethanol to match the mentioned 1/10th. I am afraid now that the DNA might have been harmed (broken etc.) by the high salt concentration at the beginning. The vector I am using is pretty big (17 kb).
Do you think I cann still use the DNA or should I toss it away?
Thanks,
Lisa
I have a question: After a Phenol/Chloroform Extraction I added too much salt: to a mix of 400ul I added 140 ul of 5M NaCl, instead of only 40 ul (1/10) as it was written in the protocol. The DNA pellet fell out immediately. After 20 min I realized my mistake and added an according amount of buffer and ethanol to match the mentioned 1/10th. I am afraid now that the DNA might have been harmed (broken etc.) by the high salt concentration at the beginning. The vector I am using is pretty big (17 kb).
Do you think I cann still use the DNA or should I toss it away?
Thanks,
Lisa
Always use it and see what happens... you will either learn that it does not work or you will know what can work... you are gaining experience both ways....
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#4
Lisa86
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Posted 21 January 2011 - 04:48 AM
Hi,
thanks for your answers! It goes directly into ES Cells, so I can not really try it and see what happens. I have no method of control before that happens, other than giving it to sequencing again....
thanks for your answers! It goes directly into ES Cells, so I can not really try it and see what happens. I have no method of control before that happens, other than giving it to sequencing again....
#5
mdfenko
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Posted 21 January 2011 - 01:11 PM
the dna should not have been harmed by the high salt. if you think it may be broken then you can run it on a gel, but that should be unnecessary.
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genius does what it must
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#6
jonas albarnaz
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Posted 13 March 2011 - 09:37 PM
Lisa,
If you will use this DNA for subsequent enzymatic reactions, such as ligation, salt excess will certainly inhibit them. I say it by my own experience, unfortunately...
Good luck in the next DNA preparation!
Best,
Jonas
If you will use this DNA for subsequent enzymatic reactions, such as ligation, salt excess will certainly inhibit them. I say it by my own experience, unfortunately...
Good luck in the next DNA preparation!
Best,
Jonas
