Double Digest problems - (Nov/03/2004 )
The problem I've been having with my digestion is one I've never seen before. When we run the Uncut pET32b+ vector (5.9Kb) along side of the double digested vector ( 5.4Kb cut with NdeI and HindIII) in a 1% Agarose gel the uncut vector shows a lower MW band than the cut vector by about 1kb. When the vector is cut with only one Restriction enzyme the band was significantly higher than the double digest and nondigested at around 8kb.
Has anyone seen this phenomenon? If so any suggestions?
Did you check the restriction map to ensure there is only one cut site for each of the enzymes?
the fact that your undigested plasmid is lower than double digest wouldn't worry me, uncut vectors run anywhere almost:) The fact that your single-digested vector runs higher though suggests to me that it's not a complete digest and your plasmid might be just getting nicked- single strand cut, which means that it'll get unwound and so tangle up wothin the gel. Make sure you have a godd site within the vector and that your digest has enough enzyme, appropriate buffer and continues for enough time.