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.
