DNA QUANTITATION

frist at ccu.umanitoba.ca frist at ccu.umanitoba.ca
Thu Oct 3 09:15:22 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.
>
.... deleted description of purification......
>-= Steve =- 	AGOODRID at VAXA.WEEG.UIOWA.EDU
>
Another way of getting rid of RNA in plasmid preps is RNAseA digestion,
followed by PEG precipitation. After the initial alkali lysis  and ensuing
steps, resuspend your EtOH pellet in 25mm Tris Cl pH8.0 30mM EDTA.
Digest with RNAseA [10ug/ml] at 37C, 15min. Add 0.4 vol. of 30% PEG6000,
1.8M NaCl (note: use MCB or EM PEG6000, not Sigma.)  Incubate 0-4C 4hr.
Centrifuge 10 min. At this point, you can either do ethanol precipitation 
or if you need really clean DNA, add a phenol:CHCl3 step as well. 

I have found this PEG precipitation step very useful in cleaning up
miniprep DNA for double-stranded sequencing. It is a good purification step
because it works on the basis of molecular weight, leaving behind small
molecules of all kinds, including oligonucleotides left over from RNAse
digestion. See Lis J.T. (1980) Meth. in Enz. 65:347-353 for more on
PEG precipitation.

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