Cant get rid of 2kb contaminant band during cloning
tvink at xs4all.nl
Sun Oct 6 06:58:03 EST 2002
Could also be an F-episome or alike present in your bacteria (eg. for
example xl1-blue or JM109) copurifying with your plasmid. When your
plasmid is low copy you will surely see any episome present in the
prep. Donot worry about it , it doesnot disturb most expreiments or
retransform in a bug without episomes eg Dh5alpha or alike.
On 5 Oct 2002 20:36:03 +0100, marsupilamiwolfgang at web.de (Wolfgang
>looks like 2kb band is supercoiled and can't be digested.
>This can happen when the alkaline lysis step in the plasmid prep is too
>long. Many kits suggest 5 min or even longer, but in the case of standard
>E. coli strains, a 1 or 2 minutes (basically the time until all bacteria
>seem to be lysed and the slurry looks homogenuous) are sufficient.
>Add SDS/NaOH, gently invert and snap the tube a few times with your
>fingers, then put on ice and immediately add the neutralizing solution
>At 21:27 04.10.2002 -0000, rpandy2000 at yahoo.com wrote:
>>clone has a 5kb insert in a 4kb vector. After mini/midi preps, however
>only a weak band at 9kb
>>observed.Major band is a 2kb one on 1% agarose gel. gel isolated 9kb band,
>>regrew up cultures...2kb band shows up again...restriction digests do not
>cut 2kb band but does
>>cut 9kb band. sequencing indicates presence of complete insert in the DNA
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