Oligo phosphorylation and annealing

1. 1. Phosphorylate the 5' end of each oligo separately by treating with PNK - let following mixture sit for 1 hr at 37C:

- 1 μl oligo (100µM)

- 1 μl 10x ligase buffer

- 7 μl H₂O

- 1μl T4 PNK

2. Mix the 10 μl of each phosphorylated oligo together and bring the total volume to 200 μl with water

3. Take 50 μl of the mixture and run it on the thermocycler using the "Anneal" program:

1. 96˚C for 6 min

2. 0.1˚C per second ramp down to 23˚C

3. Hold at 23˚C

Small fragment Golden Gate assembly

4. Use 2 μl of the mixture to ligate into the sgRNA entry vector, pWS082:

- 2 μl of annealed oligos

- 0.5 μl of sgRNA entry vector

- 1 μl 10x ligase buffer

- 1 μl BsmBI

- 1 μl T7 DNA ligase

5. Thermocylcer protocol

1. 42°C for 2min

2. 16°C for 5min

3. Repeat steps 1-2 (10x)

4. 60°C for 10min

5. 80°C for 10min

6. Transform half the reaction mixture into E.coli

7. Select non-GFP colony and inoculate in selective media

8. Miniprep plasmid and send for sequencing