electrophoresis in agarose gel

[Ulrich Maier]

Hang-Rae Kim <chamel at plaza.snu.ac.kr> wrote:

>I am handling the plasmid pIH821(6.2kb, New England Biolab) for
>expressiong the fusion protein. 
>So The plasmid was digested by restriction enzyme(NeB) EcoR1, EcoR1 site
>was including at the polylinker site.
>The digested plasmid was running the 1% agarose gel, I eluted the band
>of 6.2kb plasmid by QIAGEN kits(LRS lab).

>But when the eluted plasmid was running 1% agarose, I found the two
>bands of 6.2kb, 2-3kb. But this 2-3kb band was variable in size from 3kb
>to <2kb.
>I don't know that 2-3kb plamid band is generated from eluted plasmid
>DNA.


>I hope you your kindness.

>chamel at plaza.snu.ac.kr

Two possible explanations for the phenomenon:

I think your 2-3kb band is circular Plasmid DNA. It is possible that
small amounts of circular DNA are comigrating with the linearized
plasmid during the first gel electrophoresis.

Second trial:
You have nicked your plasmid during digestion with EcoRI. The
follow-up steps led to a DNA-fragmentation visible on the second gel.

Just a few thoughts of mine,

Ulrich Maier