DNA QUANTITATION

Chris_Upton at darwin.biochem.ualberta.ca Chris_Upton at darwin.biochem.ualberta.ca
Wed Oct 2 15:55:09 EST 1991


In article <2OCT199111074224 at vaxa.weeg.uiowa.edu> agoodrid at vaxa.weeg.uiowa.edu
(BIOCHEMISTRY) writes:
>
>muriana at aclcb.purdue.edu (murianap)  writes:
>
>>I presume that the presence of individual RNA bases would still
>>give absorbance and mask the true levels of DNA quantitation by
>>absorbance at 260nm.  Am I correct? and would this be alleviated by
>>ethanol  precipitation, whereby the digested bases would not be
>>precipitated by ethanol, so the DNA can be revovered again for quantitation.
>
>Peter,
>
>In short the answer to both questions is yes.
>
>The bases do absorb at A260.  However, I would like to say something
>about RNase digestions.  In my experience with plasmid preps only extensive
>digestions with RNase along with precipitation effectively remove RNA.
>
>Test a sample of your DNA by running a high percentage gel (eg Nuseive 5%)
>and look for RNA bases and oligos.  In my experience RNases A and T1
>(a common mix for effective removal of RNA) do not digest RNA to completion.
>The resulting oligos can also precipitate.  So when I don't use a column
>I perform two cycles of digestion and precipitation.  The purity of the
>DNA from RNA is good after this procedure but then I suppose that depends
>on what you are going to use the DNA for.
>
>I would stay away from isopropanol precipitations since it is effective
>in precipitating oligos.
>
>Hopefully, these answers are better than the ones I gave you previously.
>:-)
>
>-= Steve =-     AGOODRID at VAXA.WEEG.UIOWA.EDU
>
>
  I'm in the midst of testing a protocol to remove RNA from mini-preps for DNA
sequencing. We've just taken delivery of an ABI sequencer and the suggestion is
to get rid of RNA.
So, I added RNAse to my Soln I (of the std Rapid Alkali prep)....
1) Pellet 1.5.ml cells, remove medium
2) Add 100 microlitres soln I, vortex and leave at RT 10 mins
3) Add 200 microlitres soln II (SDS/NaOH), mix and put on ice 10 mins
4) Add 150 microlitres soln III (Na Acetate), vortex, leave on ice 10 min
5) microfuge 5 mins, tip supernatant into new tube
6) Do Glass milk purification....
7) Add 2 vols NaI soln + 20 microlitres of glass milk, vortex, on ice 10 min
8) Microfuge, remove SN, wash 3 times with EtOH/salt soln
9) Remove last SN completely and dry pellet with kimwipe
10) Resuspend pellet in 20 microlitres of water, 60 C 10 min
11) Microfuge and KEEP SN
 
DNA cuts like a charm. NO RNA bits left. I also found if I just did the 
usual P/C ppt I got lots of RNA degradation products.
Well the reactions are on the sequencer.....
 
We make our own glass milk so it costs nothing!
The process does lead to a little shearing of the DNA depending how
vigorously one vortexes during the glass milk process.
I'm hoping this will give good template.
I also scaled up the reaction to start with 5ml of culture, but just 
resuspended that in 100 microlitre of Soln I.
I'll let you know how it works.
   
Chris Upton



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