Cloning of guideRNA-Oligo into pBluescript U6-MCS

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 BbsI digested vector pBluescript_U6-MCS

• Digestion of about 4 µg pBluescript_U6-MCS with 2 µl BbsI (10 Units in NEB buffer 2.1) in a total volume of 50 µl over night at 37° C
• Purification of vector via agarose-gel electrophoresis (expected size about 3370 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 pBluescript_U6- MCS/BbsI (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), the next day 10 clones are tested via colony PCR
• Analysis via colony PCR forward primer No. 1371, reverse primer (antisense oligo of used gRNA pair),

$⟶$ positive control: plasmid No. 3761 (pBS_U6-gRNA-mParvinB), primers #1371 + #2441; PCR product of about 230 bps; negative control: plasmid No. 3468, primers #1371 and antisense oligo of used gRNA pair; no PCR product; analysis via 2% agarose gel

• Sequencing of positive clone: Mini preparation use T7 primer for sequencing