EcoRI problem - (Jan/24/2006 )
Weird things are happening here. I have a plasmid in the freezer, a qiagen-prep. When I cut this with EcoRI it just opens and I have a 6.7kb fragment. So far so good. When I want to use this plasmid in an other cloning experiment, take it out of the freezer and cut it again with EcoRI the size of the band is much bigger, now about 13kb??? It looks completely cut, I can see only one band. For the digest I used 1 microgram DNA and 10units EcoRI, left it for 2 hours at 37C. What is happening here?
WHat is the size of the vector+insert? May be its incomplete digestion. Check the restriction sites again.
Weird things are happening here. I have a plasmid in the freezer, a qiagen-prep. When I cut this with EcoRI it just opens and I have a 6.7kb fragment. So far so good. When I want to use this plasmid in an other cloning experiment, take it out of the freezer and cut it again with EcoRI the size of the band is much bigger, now about 13kb??? It looks completely cut, I can see only one band. For the digest I used 1 microgram DNA and 10units EcoRI, left it for 2 hours at 37C. What is happening here?
WHat is the size of the vector+insert? May be its incomplete digestion. Check the restriction sites again.
Until now it is just an empty vector size 6.7kb with an unique EcoRI...
i was having problems with EcoRI too but it was simlpy not digesting....maybe 10 units for 1 ug is too much? and why 2 hours i think 1 hour is enough.....myabe some star activity or something? 
Your parent vector could be two copies of the vector you think you have, in direct repeat. You could be seeing partially cut vector with two copies showing as a single 13 Kb band. Further cutting cuts the second EcoRI site and gives two linear copies of the vector you thought you had originally.
Some things to know about EcoRI:
Star activity is common at high glycerol concentrations and at high DNA concentration. Use dilute reactions (1 ug in 100ul reactions). The custom buffer (at NEB at least) contains Triton-X100. Our experiments show that there is enough carryover of this detergent to cause dramatically reduced transformation efficiency if the cut and heat-killed digested DNA is added directly to a ligation mix, ligated, and transformed. We have switched to NEB buffer 2 for this reason, although it provides less control over star activity.