Does any body had used Nsi I enzyme from MBI- Fermentas, i have used three different lot of the enzymes and in my case this enzyme is not working. I have checked it with three different plasmids also.
i have done that, they are ready replace also but they are not ready agree that it is not working
Hi Preeti
Could you give some more information about how you did the digest? How long, what was the total amount of liquid in your digest, how many units of enzyme, how much DNA, how did the (non)-digested DNA look like? Are you sure that the 3 plasmids you tested really have a recognition site (= do you have a plasmid prep with which someone else did a successful digest with NsiI before?)?
Do the enzymes arrive with dry ice or ice blocks when they arrive in your lab?
It's quite unusual and unlikely that 3 different lots of enzyme sent at 3 different dates do not work...
We also use enzymes from Fermentas, and they work pretty well.
generally i do RE digestion reaction with 10 units of enzymes, 1 microgram of plasmid, 1X buffer in total 30 to 50 microliter reaction volume at 37 degree for 4- 16 hours I have also checked the gene sequence of all plasmid i know the right size of band, you might be rihgt that it is strange that 3 lots of enzyme. I was also not ready to beleive as i am using fermentas enzymes from last 9 years. Only thing i would like to try is to test the other company's enzymes
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