Dead stab culture - what to do?

D.K. dk at no.email.thankstospam.net
Fri Mar 15 22:43:35 EST 2002


Frank at Fackelmayer.de wrote:
>Cornelius Krasel wrote:
>> 
>
>> to avoid searching all issues if possible. Therefore, if anyone can
>> give a more detailed reference or (even better) a method for
>> the purification of plasmids from stab cultures, I would be very
>> happy :-)
>> 
>
>
>hi Cornelius,
>I guess the only way is to declare the cells dead and try to recover the
>plasmid. What I did several times with old agar plates should also work
>with stabs. Take a part of the stab containing as much bacteria as
>possible, put it into an eppendorf cup, and homogenize it in GET buffer
>(solution 1 for minipreps). Continue like for a conventional miniprep,
>and spin out agar and cell debris after neutralization. Precipitate DNA
>with ethanol and use for electroporation of fresh bacteria with good
>cloning efficiency. I got several colonies from VERY old plates, and the
>DNA was ok.
>

Yep, I did this too, several times. Always worked. 

DK




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