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Smear in plasmid sample - (Oct/14/2012 )

Can anyone explain the occurrence of an oddly short smear 'band' from a plasmid sample which I recently obtained via Midiprep. Prep was done using Qiagen Midi. 1% agarose was ran for 75V for 30 min followed by 100V for around 10 min. Attached Image

-science noob-

What sort of size is that band?

-bob1-

bob1 on Mon Oct 15 07:51:19 2012 said:


What sort of size is that band?


Its a 10-comb standard well size if that's what you're asking.

-science noob-

Looks like a large plasmid and lots of DNA loaded into the slot. Try less.

-cellthetruth-

science noob on Mon Oct 15 11:38:41 2012 said:


bob1 on Mon Oct 15 07:51:19 2012 said:


What sort of size is that band?


Its a 10-comb standard well size if that's what you're asking.

I was more thinking molecular mass or basepairs...

-bob1-

bob1 on Mon Oct 15 19:25:31 2012 said:


science noob on Mon Oct 15 11:38:41 2012 said:


bob1 on Mon Oct 15 07:51:19 2012 said:


What sort of size is that band?


Its a 10-comb standard well size if that's what you're asking.

I was more thinking molecular mass or basepairs...


15-18kb

-science noob-

this is what I get when I load twice the amount of plasmid DNA to the picture I showed previously:


any insights/explanations??
Attached Image

-science noob-

My guesses would be too much DNA, and also damage to the well (maybe when you pull the comb out, or during loading). I see this kind of thing (the intense spots in the middle of the band) quite a lot in classes with undergrads when they have actually put their tip into the well to load the DNA rather than just hovering above it and letting it fall in. I think maybe some of the DNA gets "injected" into the gel itself or a gouge is taken from the wall, so it is more conc in that spot and runs through the gel that way (if that makes sense?).

Also, without knowing much about your tanks etc, I would say that running at 100V (assuming you were using TAE or something) might have the gel heating too much, so that it melts a little and this would also contribute to your bands smearing.

-leelee-

Getting good resolution on an agarose gel at 15K-18K bases is difficult or impossible. Anything over about 15Kb runs at about the same spot, and smears. You should be using a very low percentage gel, 0.5% to 0.6%. These gels are very fragile.

-phage434-

make sure your gel and buffer are not old. and try low voltage for the electrophoresis.

-gyma-