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
