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
>I don't know that 2-3kb plamid band is generated from eluted plasmid
>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.
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,