Patent ID: 8795988
Filing Date: 2014-08-05
Classification: C12N

Abstract:
1. A method for making an shRNA expression vector, comprising the steps of a. providing first and second DNA oligonucleotide primers, wherein i) said first DNA oligonucleotide primer comprises sequences encoding ii) said second DNA oligonucleotide primer comprises sequences encoding b. annealing said first RNA loop sequence and said RNA loop sequence to form an annealed structure; c. filling in single strand segments of said annealed structure to form a double-strand structure; d. cleaving said double-strand structure using restriction enzymes that recognize said first and said second restriction enzyme recognition sequences, to form a structure with a 3′ overhang and a 5′ overhang; and, e. ligating said structure with a 3′ overhang and a 5′ overhang into an expression vector to form an shRNA expression vector; and wherein said first RNA loop sequence and said second RNA loop sequence are 8-10 Nucleotides in length.