Simultaneous knockdown of the expression of two genes using multiple shRNAs and subsequent knock-in of their expression

. Author manuscript; available in PMC: 2009 Sep 28.

Published in final edited form as: Nat Protoc. 2009 Aug 27;4(9):1338–1348. doi: 10.1038/nprot.2009.145

Component	Volume per reaction (μl)	Final concentration or amount
2× Ligation buffer	10 μl	1 × reaction buffer
annealed oligonucleotides mix from step 9 (0.1 mg/ml)	1 μl	100 ng
BamH I and Hind III digested shRNA vector from step 13 (variable concentration)	variable	50 ng
Quick Ligase (2.0 × 10⁶ units/ml)	1.0 μl	2,000 units
ddH₂O	variable	To a final volume of 20 μl