I am trying to cut ~ 5ug of pUC19 plasmid, exposed to very high UVC dose (~800 J/m2) using T4 endonuclease V from NEB. Everything is being done according to NEB Protocol. I tried increasing the incubation times and units of enzyme being used in the reaction. But only ~10-20% plasmid goes from supercoiled to nicked, though 800J/m2 of UVC should induce something like 50 CPD per plasmid. Any suggestions from anybody?
Hi
Have you changed your restriction enzyme lot recently? Was it working previously? Changing the enzyme lot might help.
It never worked to begin with. NEB sent 3 more vials of same lot, none worked. I changed almost everything, no effect. I guess this particular lot might be faulty but I am having a hard time. Do you know any other good source of T4 EndoV?
Hi Vijayendra, NEB had only that particular lot at the time. But you will be surprised to know that a different amount of T4 endo packing, of the same lot is working. After sending me 3 vials of 500U, NEB sent their own positive control plasmid along with a new vial of T4 endo, same lot number but 1000U packing. This vial is cutting NEB positive control plasmid, my own UV exposed control plasmid and genomic DNA too, using all the same conditions what I was using earlier. NEB is not able to explain, neither am I and my boss, that why should this happen? BUT it is working now. I will be careful now about buying this enzyme from NEB..
Great Sanjay! Finally you got the things rolling. I am equally surprised by your experience.
- Badges
- Science topic
- Similar topics
- Cell Biology
- Methods