5 replies to this topic

[Wek]

What is the absolute max. time for a 2cm tail snip digestion with proteinase k? I usually digest it overnight and it is fine but I was wondering what would happen to the DNA (if anything) if the tail is left digestion for 2-3 days at 55C. Will proteinase K affect DNA?

[prabhubct]

Long time digestion may not interfere with DNA as all DNases are supposed to be inactivated, though as per http://www.piercenet...?fldID=06010216 they recommend short time of digestion. may be they have standardized time. Why you want to waste your time reinventing wheel?

[bob1]

There is potentially the issue of degradation of the DNA through acid hydrolysis, but this shouldn't be too much of a problem if you are using a buffered system.

[Wek]

prabhubct, on 27 October 2012 - 10:01 PM, said:

Long time digestion may not interfere with DNA as all DNases are supposed to be inactivated, though as per http://www.piercenet...?fldID=06010216 they recommend short time of digestion. may be they have standardized time. Why you want to waste your time reinventing wheel?

I left some samples to digest overnight on Friday but am not able to work on them until Monday or Tuesday. Not sure if my colleague received my text about inactivating the enzyme and putting them in 4C.

bob1, on 27 October 2012 - 10:36 PM, said:

There is potentially the issue of degradation of the DNA through acid hydrolysis, but this shouldn't be too much of a problem if you are using a buffered system.

Samples are in TNES buffer so I guess it isn't going to be a problem.

How stable is DNA at RT and at 55C while in TNES?

[bob1]

The higner the temperature the less stable it is.  I am not aware of any studies looking at the stability in TNES specifically, but there is a study comparing DNA stored in water, tris alone and TE.  Basically the conclusion is that DNA is most stable in TE and frozen, but will keep for an extended period of time at fridge temps.  Room temp resulted in some degradation over a week or two.

[prabhubct]

55⁰C that look like annealing temperature for 3 days. I guess you could try process it. It may remain stable.

have look @ https://brd.nci.nih....d=11966&cid=727

Edited by prabhubct, 28 October 2012 - 01:48 AM.