digested run slower on gel? - (Feb/13/2008 )
Is the restriction enzyme digested plasmid run slower than the undigested? agarose gel(1%))
Hmm, can you specify what you observed? 
I may misunderstand you but maybe you observed different conformations of your plasmid DNA: single cut linearises your plasmid and this conformation runs differently than the relaxed circular/closed circular/supercoiled conformation of your undigested plasmid. Any help?
(Digest at more than one position of your plasmid should result in DNA fragments, that are smaller than your original plasmid and therefore run faster on a gel though.)
yeah
I used bamh and NdeI to digest my plasmid, so the digested one should have less bps than the undigested one, so it should run faster, but the result is the opposite.
so confused...
supercoiled plasmid will migrate faster than its size would indicate. when you cut it you relax the supercoil and it will run truer.
were you cutting with both restriction enzymes at the same time (same batch of plasmid) or separately (different batches of plasmid)?
if separately then you are linearizing the plasmid and relaxing the supercoil.
if together then you should see 2 bands (or more if incomplete digestion).
If you could post the gel pic, it might be easier to explain things.
What are the sizes of the fragments and your original plasmid?
sorry i didn't take a pic then
and they were cut with both enzymes at the same time
the vector is pet20b which has around 3700 bps and the sequence between two restriction enzyme should be around 80
and they were cut with both enzymes at the same time
the vector is pet20b which has around 3700 bps and the sequence between two restriction enzyme should be around 80
As mdfenko mentioned, you probably see supercoiled vector in the lane with undigested product, which runs faster and thereby lower in the gel than the rest vector in its linearised form (according to your description only 80 bp smaller) in the lane with the digested product. You may want to include single cut controls when you repeat the experiment - this should linearise your vector resulting in a band similar to the one you have seen in the double cut (confirming differences between your samples being due to different conformation of the plasmid).