No band on double digestion of construct - (Feb/09/2006 )

Hi!

I have extracted my recombinant plasmid with contain 2886bp of vector and 2510bp of insert.
I did double digest using BamHI and EcoRI with 2X TAngo buffer by Fermentas.

The recipe as below
10X buffer 10microL
R. plasmid 5micro L (5 microG)
Bam HI 10 micro L (10U)
EcoRI 5 microL (5U)
Water 20 microL

Total volume = 50 micro L

Bam HI has 50-100% activity and EcoRI has 100% acitivity. thats why I double the amount of BamHI


After incubation at 37C for 1 hour, I run the gel. There was no band at all in the gel, only the DNA markers. What could have happen. Anything wrong with my recipe?

15µl of enzymes in 50µl final????? 10x buffer 10µl??????
the glycerol conc will inhibits the digestion....
come on

1µl of each enzyme in 50µl 3h is enough for 5µg...