Linearized vector runs as two bands?

Frederik Wirtz-Peitz fw218 at cam.ac.uk
Mon Jun 11 06:24:46 EST 2001


I'm wondering if anyone can offer an explanation for the unusual migration
of linearized vector in some of my prep gels (0.8% agarose). There is a main
band (about 75% of the total DNA) running at the expected position as well
as a secondary band of seemingly higher molecular weight. I couldn't
determine the exact mass because I was using small markers, but I suppose it
came in at roughly twice the mass of the vector.

The only explanation I can come up with is that the linearized vector
concatamerized. But since the enzyme I was using produces only 4 bp
overhangs, I'm tempted to discount that possibility. Still, could this
possibility be excluded by heating the sample subsequent to digestion? If
so, what temperature?

It may be worth noting that I've had this problem only with derivatives of a
certain vector (but not the vector itself!). I subcloned two completely
unrelated inserts into this vector, and both derivatives exhibited the
phenomenon. I subcloned some more stuff into these derivatives, and the
unusual banding persisted. The size of these plasmids was in the range of
6-7 kb. At the same time I was linearizing other vectors (using the same
reagents), and all of those migrated as a single band.

I don't know how common this phenomenon is. I'm mainly interested out of
curiosity. But at the time it was also cutting down on my yields!

Thanks in advance.





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