No banding in CsCl Spin,Why?

Vlad Svetlov vvsvetlov at utmem1.utmem.edu
Mon Aug 12 03:20:27 EST 1996


In article <1.5.4.32.19960812110810.006649f0 at pop3.jaring.my>,
spyong at pc.jaring.my (Stephanie Yong) wrote:

> Hello,
> 
> I tried to purify my plasmid through CsCl centrifugation, and there was no
> band.I used the Alkaline Lysis method using Solution 1,followed by Solution
> 2 and 3.Then I added 1g/1ml of CsCl into the lysate with Ethidium Bromide

Unless Bible has an Alkaline lysis protocol there are no single protocol
people use and purity of your DNA prep (and thus its behaviour during
centrifugation) depends on what particular method was used. If you did not
use RNase and proteinase K during isolation your sup (I assume you did do
spinning after sol 3) is full of RNA and protein junk which often affect
appearance of the bands. You'll be much better off precipitating (better
couple of times) your DNA with isopropanol before adding CsCl - if you got
to work with a lot of EtBr it's not worth saving few hours on
precipitation/resuspension. Pretreating your stuff with RNase and
proteinase K (successively) should also help reduce that poetically
described in Maniatis "furry scum that floats to the top" (scum always
does, doesn't it?). 
Regards,
V.



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