Cloning of guideRNA-Oligo into pLentiCRISPRv2

Materials

gRNA Oligos (sense/antisense; designed using ChopChop)
Vector pBluescript_U6-MCS digested with BbsI (Plasmid #3468)
T4 DNA ligase; 10x Ligase buffer
E. coli NovaBlue

Procedure

gRNA Oligo annealing

• annealing reaction contains: 1 µl sense primer (stock solution 100 µM), 1 µl antisense primer (stock solution 100 µM), 5 µl NEB buffer 2 and 43 µl ddH₂O
• Annealing reaction occurs in thermocycler with the corresponding program:

95°C	4 minutes
Start loop, 54x, 94°C, 1 min (-1°C/loop)
8°C	Store forever