Cloning of shRNA into lentiviral vector pLKO.1

Procedure

Primer 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:

1	Hot start	94°C	4 minutes
Start loop, 24 cycles
2		94°C	1 minutes (-1°C/min)
3	Close loop
4		70°C	10 minutes
Start loop, ?? cycles
5		70°C	1 min (-0.1°C/min down to 4°C)
6	Close loop
7	Store forever	4°C

Preparation of the lentiviral vector pLKO.1

• Double digestion of about 5 µg pLKO.1 with AgeI and EcoRI (in NEB buffer 1) over night at 37° C
• Dephosphorylation of vector: 3-5 µg DNA, 1 µl phosphatase, 1/10 vol 10x buffer; incubation for 30 min at 37° C; heat inactivation for 10 min at 65°C
• Purification of vector via agarose-gelelectrophoresis (expected size about 7000 bps; original size about 8900 bps)

Ligation, Transformation and Analysis of Clones

• Ligation reaction of vector and insert contains: 1 µl digested pLKO.1 (about 100 ng), 1 µl annealed primer, 1 µl T4 ligase, 1µl T4 ligase buffer and 6 µl ddH₂O; incubation for 4 h at 4° C
• Transformation in E.coli nova blue (plating on LB- Amp), the next day clones are picked and expanded on a new plate
• Mini preparation (Birnboim-dooley) and subsequent analysis via agarose- gelelectrophoresis (expected size about 7000 bps, positive control plasmid No. 2512)
• Analysis of potential positive clones via PCR: 1 µl of 1:10 diluted mini preparation, 1 µl forward primer (No. 1371), 1 µl reverse primer (No. 1372), 1 µl dNTPs, 5 µl 10x Taq-buffer, 1 µl Taq-polymerase and 40 µl ddH₂O;
• Positive control: plasmid No. 2512, PCR product of about 250 bps; negative control: plasmid No. 2521, PCR product of about 190 bps; analysis via 2% agarosegel
• Sequencing of positive clone: Mini preparation (Qiagen; elution with EB); 1:10 dilution of primer No.1438 in EB