GuideRNA

1. Annealing Buffer

• a. 400 μl 1M Tris @ pH 8
• b. 200 μl MgCl2
• c. 100 μl 5M NaCl
• d. 20 μl 0.5M EDTA @ pH8
• e. 280 μl H2O

2. BsaI-HF
3. NEB 4
4. Ligase
5. QLB
6. Kanamycin
7. XL1-Blue chemically competent cells.

Steps:
1. Digest backbone (pDR274)

• a. 1 μg of pDR274
• b. 5 μl BsaI-HF
• c. 5 μl NEB 4
• d. H2O to 50 μl

2. Gel isolate digested backbone
3. Normalize to 5 ng/ μl
4. Anneal Oligos in thermocycler

• a. 100 μM Oligo 1
• b. 100 μM Oligo 2
• c. 5 μl 10X annealing buffer
• d. 43 μl H2O
• e. 95 °C
• f. -1°C per 30 seconds
• g. 4 °C

5. Ligate

• a.1 μl backbone (@ 5ng/μl)
• b.3 μl annealed oligo
• c.1 μl ligase
• d. 5 μl QLB

15 Min at room temp

6. Transform

• a. 5 μl of ligation
• b. 45 μl of chemically competent XL1-Blue cells
• c. Ice for 2 min
• d. 42 ° C for 45 seconds
• e. Ice for 2 min
• f. Add 450 μl of LB and recover at 37°C with shaking for 1 hr
  
• g. Plate 100 μl on Kanamycin plates
• h. Grow overnight at 37°C

7.Pick single colonies inoculate 1ml of LB supplemented with Kanamycin
8.Grow overnight at 37°C
9.Mini Prep
10. Sequence using M13 primer