Restriction double digest - (Dec/07/2014 )

Hello everyone,

I want to subclone my gene into pet22b vector. I did double digestion of both vector and insert plasmid with Nde1 and HindIII 1ul each in 50ul reaction with buffer 2 and incubated for 3 hours at 37degree.  Also as controls i digested my samples with individual enzymes also. After agarose, i found out that the vector was linearized and the band was moved upward than the uncut vector. The problem is with my insert plasmid. With individual enzymes, i am getting a single band of linearized plasmid. However, with double digestion, 3 bands are seen which concludes that the plasmid has not been digested. Everytime i get different results with double digestion. i think enzymes are working fine.. Can any1 please shed some light on this?? Thank you

What is your setup for the restriction digest that is not working? (How much volume of each of the components)

A common problem is inhibition by impurities in the minipreps of plasmid DNA. This happens most often when the major volume of the digestion is dilute DNA solutions, rather than water.