From owner-diagnostics@net.bio.net Tue Aug 12 23:00:00 1997 Path: biosci!biosci!not-for-mail From: Ulrich Maier Newsgroups: bionet.diagnostics Subject: Re: electrophoresis in agarose gel Date: 13 Aug 1997 00:38:06 -0700 Organization: Westfaelische Wilhelms-Universitaet Muenster, Germany Lines: 34 Sender: daemon@net.bio.net Approved: odonnell@sasa.gov.uk Distribution: world Message-ID: <5sro8u$7df@net.bio.net> NNTP-Posting-Host: net.bio.net Hang-Rae Kim 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@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 .