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

Preparation of BsmBI digested vector pLentiCRISPRv2

• Digestion of about 4 µg pLentiCRISPRv2 with 2 µl BsmBI in a total volume of 50 µl over night at 37° C
• Purification of vector via agarose-gel electrophoresis (expected size about 13000 bps) and Gel extraction kit. Elute in 50 µl EB buffer.
• Check amount of purified, digested vector on agarose gel. Freeze down 1 µl aliquots for future ligations.

Ligation, Transformation and Analysis of Clones

• Ligation of vector and annealed oligos: 1 µl digested pLEntiCRISPRv2 (about 100 ng), 1 µl annealed gRNA oligo pair, 1 µl T4 ligase, 1µl T4 ligase buffer and 6 µl ddH₂O; incubation overnight at 4° C. (Neg. control: ligation reaction of vector only w/o annealed oligo pair)
• Transformation in E.coli NovaBlue (plating on LB- Amp), Grow on 30°C ! the next day 10 clones are tested via colony PCR
• Analysis via colony PCR forward primer (sense oligo of used gRNA pair), reverse primer #2708, product: 530bp
• positive control: plasmid No. 4300 (pLentiCRISPRv2 sgCEACAM3.1), primers #2710 + #2708; PCR product of about 530 bps;
• negative control: plasmid No. 3777, primers #2708 and sense oligo of used gRNA pair; no PCR product
• analysis via 2% agarose gel
• Sequencing of positive clone: Mini preparation use T7 primer for sequencing
• subclone into E.coli Stbl4: Grow on 37°C !