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Plasmid doubled in size??

Plasmid doubled in size?? - Molecular Cloning Forum

Plasmid doubled in size?? - DNA cloning forum. Discuss the cloning of plasmids, vectors, DNA, RNA, cDNA, proteins, and other molecules for expression, sub-cloning, and other methods. Also discuss digestion, ligation, transformation, plasmid preparation, cDNA library screening, and competent cells here.


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Old 04-14-2011, 05:52 AM
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Default Plasmid doubled in size??



Hi

I'm working with a pET vector that should be 6.1 kb with my insert. Initially, I had some trouble with our cloning strains (XL1 blues) and was running out of plasmid, so ended up transforming BL21 DE3 pLysS cells. I miniprepped from those but am now seeing something strange. When analysed on a 1% agarose gel, I get 6 bands (way more than I'd expect from supercoiling) and, when linearised, shows a size of between 10-12kb.

Is is possible that the plasmid was somehow recombined by the BL21 cells? Why is it double the size it should be?
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Old 06-24-2011, 08:54 PM
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Default Re: Plasmid doubled in size??

Hi,
I believe that some strains of bacteria used for protein expression (BL21 is one I think) have a high plasmid DNA mutation rate. I know that the strain I use (A BL21 derivative) is not recommended for doing cloning in so I only transform the plasmid right before I do the protein expression experiment.

So it's possible that the bugs have scrambled your plasmid a bit. Does the protein expression still work?
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Old 08-11-2011, 04:35 AM
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Default Re: Plasmid doubled in size??

BL21(DE3) expression host contains a phage construct that expresses T7 RNA polymerase under the control of a lacUV5 promoter. Most likely you have isolated the phage instead of your plasmid.
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Old 08-13-2011, 01:42 PM
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Default Re: Plasmid doubled in size??

better use dh5 alpha
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Old 08-15-2011, 12:42 AM
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Default Re: Plasmid doubled in size??

alternatively, u can use BL21 cloning host, XL1-blue, NovaBlue or Top10 cells.
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Old 06-18-2012, 06:41 AM
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Default Re: Plasmid doubled in size??

thanks
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