rna removal from plasmid preps with Li Cl precipitation

Nick C.K. Heng hengnck at sanger.otago.ac.nz
Tue Nov 19 21:19:05 EST 1996


In article <3291EBFD.6A9A at lacdr.leidenuniv.nl>
santbrin at CHEM.LEIDENUNIV.NL (Your Personal Name) writes:

> 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

Hello, Peter.

I also use a alkaline lysis method followed by LiCl treatment. All I
know is that the LiCl removes a lot of the *high molecular weight*
stuff and perhaps some chromosomal DNA. There is still some RNA left
after treatment but a small fraction of the initial amount. I don't
know about proteins though.

I use endA strains (DH5a, XL1Blue) for my cloning experiments so I
don't have problems with endonucleases chomping up the DNA. I do use
RNAse to cleanup the residual low molecular weight RNA which is still
visible after LiCl/ethanol precipitation.

If you work with DH5alpha, the alkaline lysis method + LiCl cleanup
gives you DNA pure enough to get 500-700 bp of reliable sequence
without phenol extractions to remove the proteins/RNAse.

The value of 70ug is very high - I get about 20-50 ug from a 50 ml TB
culture and that's pUC-type copy number. I would recommend RNAse
treatment - it doesn't affect restriction endonuclease activity (not in
my experience).

Regards, Nick.



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