I've been reading this forum for advice and you are all very helpful, so maybe you can shed some light on this problem. When I do a single NdeI digest on one vector in particular, rather than seeing the expected shift upwards on a gel for the linearized, cut plasmid, I instead see two bands develop. One band is lower than the original, uncut plasmid; and the other band may be slightly higher than the uncut plasmid. With XmaI single digestion, one band appears over time that is lower than the uncut plasmid. Has anyone seen this before? In case it matters, the vector is pIVEX and it is intended for use in cell-free systems.
Looking forward to your comments, thank you!
Single-digested vector shifts downward on gel
1 reply to this topic
Posted 19 May 2011 - 12:14 PM
Hey just in case anyone else has read this and was wondering, I think I figured out the problem. The vector sample, which was given to me by another scientist, is just extremely dirty and most of it is circularized/nicked rather than supercoiled. So when I do a single digest on it, the linearized DNA runs faster on the gel as expected.