I ligated a DNA size of 3.8kb into topo TA vector. I expected a band size of about 7.7kb from my agarose gel electrophoresis after plasmid purification but got a band size of about 5kb. Pls what does this band size signify or is topo vector less than 3kb?
TOPO TA vector is apprx 3.9 Kb. Did you perform restriction digestion before running the plasmid on the gel? Perhaps there were multiple restriction sites and you missed the smaller fragments?
Hi,
Have you loaded the isolated plasmid DNA onto the agarose gel? The observed fragment size should be smaller than the actual size of the plasmid due to the plasmid conformation. You could digest the plasmid with a restriction enzyme to see the real size on the agarose gel.
Best,
Boas
Thanks all,
I did a 15mins quick digest with EcoR1 which cuts the plasmids just close to both ends of my insert. And I had checked carefully there was no EcoR1 anywhere else, neither on the insert nor the vector
Hi again,
Cutting with EcoRI is not useful. You could not distinguish between insert and backbone (3.8 and 3.9 kb). Therefore, it would be better to use an enzymes which cuts only once. One could speculate that your restriction digest is incomplete and you are looking at the plasmid in super coiled conformation. You could try separate restriction digests with different enzymes or PCR on the plasmid. Moreover, it might be helpful to digest your samples for a longer time.
Thanks so much Boas. I like to cut once on the insert, but all the restriction enzymes on the insert are also present on the vector. Will also try digesting for a longer time, though the protocol says 15mins.
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