Nucleases and DNA isolation - (Mar/27/2006 )
Hi everyone,
I had a quick question regarding nucleases (DNA and/or RNA). I was wondering if anyone knew when nucleases become active because if I were to obtain a tissue sample, do the nucleases (which are located where? cytoplasm?) become active as soon as I obtain the sample or is it when the cells are lysed?
Thanks a bunch!
I think lysis is not required; it is when the temperature comes up and the ubiquitous enzymes find their 'happy thoughts' again and are able to resume activity
could you please give more specifics about what it is you are doing with the tissue? we may be able to give you some more specific help then
Hi,
So, I am fin clipping an adult zebrafish and attempting to isolate genomic DNA from the fin tissue. I would normally clip, then rinse with 1X PBS and then place in an eppendorf with Lysis Buffer (NP-40, Tween, EDTA), heat to 98C for several minutes, add Proteinase K and then incubate at 55C. I had fin clipped a fish today and then placed the fin in Lysis Buffer and left it at room temperature for ~20min and was wondering if and how much degradation can be expected in this case. The events listed above are in chronological order, so I was wondering if much degradation can be expected since the Proteinase K had not yet been added and the fin clip was sitting in lysis buffer for 20 min.
Hope that helps, and thank you for your timely response!
So, I am fin clipping an adult zebrafish and attempting to isolate genomic DNA from the fin tissue. I would normally clip, then rinse with 1X PBS and then place in an eppendorf with Lysis Buffer (NP-40, Tween, EDTA), heat to 98C for several minutes, add Proteinase K and then incubate at 55C. I had fin clipped a fish today and then placed the fin in Lysis Buffer and left it at room temperature for ~20min and was wondering if and how much degradation can be expected in this case. The events listed above are in chronological order, so I was wondering if much degradation can be expected since the Proteinase K had not yet been added and the fin clip was sitting in lysis buffer for 20 min.
Hope that helps, and thank you for your timely response!
And the results? As I learned, normally EDTA in lysis buffer removes the cationes necessary for nuclease activity, so it should work...
hobglobin
Good point! I've been stuck behind the bench too long to be thinking clearly anymore.
Thanks a bunch.
Out of interest: what is the incubation at 98C for. You want to denature the DNA before isolation...
I have wondered this myself for quite some time and I have yet to find the answer. It does not make sense to me why I would have incubate at 98 C in lysis buffer then follow up with incubation on ice then the overnight at 55C. Any reason you can think of? I am trying to isolate genomic DNA, I do not want it to be denatured... Does lysis buffer need to be at 98 in order to work? All the protocols I have seen said to incubate at 98 for 5 min.
Also, I had a rather bad 260/280 ratio of 1.63 so I decided to re-extract proteins with another phenol/chlorofom/isoamyl procedure, and I re-spec'd the DNA and the 260/280 ratio was worse this time around at 1.45. Where would these extra proteins be coming from? Does that make sense to you?
Thanks a whole bunch!