rna removal from plasmid preps with Li Cl precipitation

Your Personal Name santbrin at CHEM.LEIDENUNIV.NL
Tue Nov 19 03:19:20 EST 1996


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
in Maniatis.
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
The Netherlands
E-mail: santbrin at lacdr.leidenUniv.nl



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