Ethanol Precipitation - added too much salt - (Jan/20/2011 )
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 think is ok to use... what is your application?
Lisa86 on Fri Jan 21 03:04:10 2011 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
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....
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....
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.
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