2 replies to this topic

[littlecell]

hello, everybody.
i have tried three different kits for mini-prep plasmid DNA purification. but i could not get rid of the chrosomal DNA contamination though i strictly followed the manual of each producer.
could anyone give me any advice? thanks in advance.

[methylnick]

Most kits use the alkali lysis method to isolate plasmid DNA.  Addition of potassium acetate prepiptates chromosomal DNA and other cellular debris so the chromosomal DNA is removed at this stage. The lysate is cleared by centrifugation and then the supernatant is usually loaded onto a column.

I have used several mini-prep kits for automated DNA sequencing and had no problem in obtaining very clean results.

The kits used were from Promega (Wizard SV), Sigma, Qiagen and Invitrogen, all giving equivalent results because they are all essentially the same kit.

If chromosomal DNA is a problem I would maybe treat the plasmid with an exonuclease to remove the DNA, the plasmid would be protected from the exonuclease because it's circular.


Nick

[littlecell]

thanks a lot. i will use the kit you recommended.