Hi...so, basically, I would say your cloning worked well. I'm wondering why the third control plasmid is a bit smaller than first and second. Are these Plasmid from three different clones? And how about the digested Plasmids...does the digested Plasmid one belongs to undigested (control) one, and so on?
Every digested Plasmid shows a fragment of ~ 500 kb which fits to your information of the first post.
Considering multiple bands: In general I would say that all except No. 3 (with two bands) are not completely digested since I don not exactly know if control three and digested three are the same. On the other side the fragment of No. 3 is a bit smaller than the others.
So I would take No 3 and one of the others and sequence them to make sure everything is right.
Now some last words to your restriction protocol...
400 ng are sometimes not enough...the smaller the fragment the more difficult it is to see the band in the gel. I once had to deal with that...I couldn't see my insert (it was ~ 300 kb) and then I ran a PCR and I got it. Since then I always try to use at least 500 ng, better 600. Usually the companies supply a 10x buffer so 2 µl of buffer would be too much in a 10 µl reaction and 1 µl enzyme is also too much. It can cause unspecific cuts, especially with EcoRI.
A good set up would be (in my opinion) for your DNA:
6 µl DNA (à 100 ng/µl)
2 µl EcoRI-buffer (10x)
0.5 µl EcoRI
11.5 µl water
--> 37°C for 1-2 h (or less if it is a fast digest enzyme)
Then load as much as possible onto the gel.
Edited by Papaver, 30 June 2012 - 04:40 AM.