Construction of U6 - based sgRNA expression vectors
Order a pair of U6 vector - specific , 23nt targeting primers for each target For each target ( GN19 ) , order a forward primer TTCGN19 for the U6 promoter vectors ; order a reverse primer AAACN19 .
Note that the N19 in the reverse primers is reverse complementary to that in the forward primer .
To order primers compatible with both the pU6x - sgRNA vectors and pT7 - gRNA ( Add gene plasmid # 4675 ; ( JAO et al 2013 ) ) , order degenerate primers WTMGGN18 ( forward ) and AMMCN18C ( reverse ) , where M = A or C , W = A or T .
Again , the N18 in the reverse primers is reverse complementary to that in the forward primer .
Mix 1µl 100µM stock each in a 20µl 1x NEB buffer 2 .
Incubate the mixture as follows : 95 °C for 5 min , ramp down to 50 °C at 0.1 °C / sec , 50 °C for 10 min chill to 4 °C at normal ramp speed .
Mix the following components to ligate the annealed oligos to the U6 vector of choice ( pU6x - sgRNA # 1 - # 5 , see diagrams and plasmid list in the Guidelines ) .
Incubate at 37 °C for 20 min .
Incubate at 16 °C for 15 min .
Incubate at 37 °C for 10 min .
Incubate at 55 °C for 15 min .
Incubate at 80 °C for 15 min ( optional ) .
The reaction is ready for transformation ( use 2 µl of the ligation and plate 10 % ofthe transformants ) .
Transform and spread onto spectinomycin ( 50µg / ml ) plates .
