BamH1-Xho1 double digestion prob - Plasmid double digestion shows 4 bands??? (Sep/06/2007 )

I want to clone a 400bp fragment into 10kb vector so I am using NEB restriction enzymes for vector and insert double digestion, namely, BamH1 and XhoI. I performed double digestion of the 10kb vector as follows-
BSA 10X- 5microL
Plasmid(10kb) 2 micro i.e. 1.2microg
DW-36microL
Buffer#2-5microl
BamH1-1microL
Xho1-1micro

After incubation at 37 degrees for 2hrs, I ran 1% gel for an hour and stained with ethidium bromide(2microg/ml). Surprisingly, I saw 4 bands- 2 of each are closely located in the digested sample of plasmid. I am wondering if there was something wrong with this. Do anyone has some experience with this enzyme combination and suggestions???

waiting for reply.. thanks

What vector are you using?

may you attach gel picture ?
POssibility of non complete digestion ?

I am using a phage derived vector

I also had a similar problem. My enzymes were BamH1, XbaI.
I read that Bam has star activity. So maybe reducing the enzyme amount or the incubation time could solve the problem.

thanks , can you tell me what was your reaction mix, later on I also need to do double digestion with BAm and Xba

if you have up to 2µg of vector, use 0.5µl of each enzyme should do the job in a 50µl reaction mixture, with buffer 2 and BSA.
I would advice to do 2h with XbaI and add then BamHI for 3h more (till with xba I)

ps : care about the fact there is a Methylation Sensitivity:
dam methylation: Blocked by overlapping