2 replies to this topic

[aidaelshaer]

i extracted CMV-pbk plasmid from my transformed bacteria.
but when i run in the presence of the original plasmid sample which i made transfomation from it ,
i found a difference ,
all samples are not digested, the plasmid is as it is.
first lane is original plasmid (isolated from more than year)
second and fourth lane are the same plasmid  isolated from jm109
third lane is the same plasmid but extracted from xl blue strain(transformed in another day)
all must be CMV-pbk
note: when i subculture in a liquid medium for plasmid isolation , i did not  add the antibiotic.
So i am very worried.
how can i be sure from my result???
and can anybody describe for me this picture?
Aida

Attached Thumbnails

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Edited by aidaelshaer, 06 May 2010 - 03:41 PM.

[bob1]

Presuming that the original sample is the one at the bottom of the picture...

Your other samples appear to be degraded.  From a plasmid isolation, you will most likely have 3 bands - supercoiled, nicked and linear.  Nicked and linear will run at defined rates on a gel, but supercoiled can run bigger or smaller than expected size.  If you get bands other than these, you either have bacterial genomic DNA or RNA contamination, or degraded plasmid.

You definitely need to add antibiotic for isolating plasmids, otherwise the plasmid is likely to be lost/bacteria containing the plasmid will get out-competed.

[HomeBrew]

It's inconclusive, in my opinion.  You need to digest the plasmids to compare them -- preps of different age and from different hosts are likely to show varying amounts of different bands in coiled form, and have some differences in form.

Most plasmids are relatively stable -- you'll not lose them to any appreciable amount if they're cultured without antibiotic for a single culture -- after all, plasmids are maintained in their hosts in the wild for extended periods (years?) without exposure to antibiotics.