stability of plasmid

John Thompson jrt at home.com
Wed Aug 11 20:39:03 EST 1999


Terry Lau <tcklau at yahoo.com> wrote:

>Hi,
>
>Recently, I clone a insert(about 2.5k) by T/A cloning and I would like
>to cut the insert from the T/A vector. I inoculate the cell from the
>glycerol stock and grow in LB-AMP broth with shaking overnight. After
>miniprep and restriction digestion, I only obtain a small smear on the
>gel. Therefore, , I should retransform the plasmid every time. I don.t
>know what is the problem. Please give me any suggestion.
>
>Thanks a lot!

Sounds like DNAse contamination to me to.  But, unless your a real
slob, it's probably in the DNA, not the restriction buffers. Some
E.coli hosts are more problematic this way. Put the DNA in restriction
buffer without enzymes to see if still smears out.  Phenol
Extraction/EtOH precipitation should clean it up if it's in your DNA.

Regards,
John Thompson



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