DNA QUANTITATION

frist at ccu.umanitoba.ca frist at ccu.umanitoba.ca
Mon Oct 7 10:33:58 EST 1991


In article <1991Oct5.184722.7828 at mnemosyne.cs.du.edu> cenderli at isis.UUCP (C. S. Enderlin) writes:
>In article <1991Oct3.141522.18804 at ccu.umanitoba.ca> frist at ccu.umanitoba.ca writes:
>>>
>>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.)  
>                                         ^^^^^^^^^
>
>Is the Sigma PEG problem peculiar to double strand sequencing?
>
>I have used Sigma PEG for ssDNA preps ad nauseam with great results.


I was trying to make a long story short, but I guess I'd better explain.
Years ago, Sigma sold a product called PEG6000, which at least in my hands,
resulted in incomplete restriction of plasmid DNA when used in the prep. It
turned out that after more careful assay, the avg. molecular weight of that
product was closer to 8000 than 6000, and Sigma has subsequently re-labeled
their product. In the meantime, I found that using PEG 6000 (really
6000-7500) from MCB or EM gave much better results, and I have stuck with
them ever since.

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