No banding in CsCl Spin,Why?

brett brett at BORCIM.WUSTL.EDU
Mon Aug 12 13:38:06 EST 1996


>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
>and was set to spin in a 15ml ultracentrifuge tube.I used a  Sorvall
>vertical rotor at 45,000rpm for 16hrs at 21 degree Celcius.
>
>Should I precipitate after Solution 3,then resuspend in TE first before
>adding CsCl?
>
>I though my broth was contaminated and it wasn't my  E.Coli containing my
>plasmid that was growing,but,I've double checked my plasmid through a
>miniprep and it is there,no contamination.
>
>Please advise.
>
>My E-mail is : spyong at pc.jaring.my
>
>Thank you,
>Stephanie.

Precipitate your plasmid, then resuspend it. As a shortcut to weighing out and
dissolving CsCl for every sample I do, I resuspend in 1.7ml TE, add 3.2ml of
a saturated CsCl solution (25*C), and add EtBr. Final volume ca. 5ml. Spin
in Ti90 near-vertical @ 65krpm for 4hrs., then pull out my bands. Good luck.



Brett Lindenbach
    
Program in Immunology                              
Washington University - St Louis                  
brett at borcim.wustl.edu                             

"I own my own pet virus. I get to pet and name her." - Cobain




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