Cant get rid of 2kb contaminant band during cloning

Tom Vink 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.

grtz Tom


On 5 Oct 2002 20:36:03 +0100, marsupilamiwolfgang at web.de (Wolfgang
Schechinger) wrote:

>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
>(acetate).
>
>Wo
>
>
>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,
>transformed and 
>>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
>prep...HELP 
>>
>>http://biowww.net/mynews/tree.php?group_name=bionet_molbio_methds-reagnts&b
>egin=0
>>
>>
>
>---




More information about the Methods mailing list