Cant get rid of 2kb contaminant band during cloning

Wolfgang Schechinger marsupilamiwolfgang at web.de
Sat Oct 5 14:36:03 EST 2002


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
>
>

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