rna removal from plasmid preps with Li Cl precipitation
weber at cmb.UNR.EDU
Tue Nov 19 16:18:36 EST 1996
In article <3291EBFD.6A9A at lacdr.leidenuniv.nl> santbrin at CHEM.LEIDENUNIV.NL (Your Personal Name) writes:
>From: santbrin at CHEM.LEIDENUNIV.NL (Your Personal Name)
>Subject: rna removal from plasmid preps with Li Cl precipitation
>Date: 19 Nov 1996 00:19:20 -0800
>To isolate my plasmid I used the QIAGEN colums but
>because the are so expensive I stop after the P3-step
>(addition of 3 M Kac, pH 5,5) and precipitate with
>isopropanol followed by phenol/chloroform and chloroform
>extraction to remove proteins (esspecially endonucleases)
>To remove RNA I precipitate with LiCl following the instructions
>My questions are:
> - must the RNA be "intact" (isolate without RNase treatment) to
> have an efficient purification?
> - does this method remove all the RNA?
>I ask this last question because when I looked at a fraction on
>agarose gel it looked clean, but when I measured the A260 I isolated
>about 70 ug from a 100 ul (that is microliters) O/N TB-culture, wich
>seems to be a bit high, don't you think? The A260/A280 was 1.9.
>I really like the method because it saves us a lot of money but I
>need this plasmid both for transfection and sequence experiments and
>RNA polution can give problems.
>Can anybody give me an answer to my problems? Thanks in advance
>Peter van Santbrink
>University of Leiden
High MW RNA greater than about 100 nucleotides precipitates from high salt in
the cold. DNA does not. The use of LiCl goes back to the early 70's where it
was used to precipitate high MW RNA in the presence of SDS in the cold.
Lithium dodecyl sulfate is soluable in the cold. There is nothing special
about LiCl. 2.5 M ammonium acetate works just as well for plasmid preps.
Both methods leave tRNA and small RNA fragments in the supernatant, but remove
about 90% of the total RNA as well as most residual protein impurities. Don't
treat with RNase or the method will not work. The small amount of low MW RNA
often does not interfere with sequening or transfection . But sometime it
does. You just have to test this our for yourself vs more highly purified
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