Paul.xu: Created page with "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. QL..."

2013-05-14T17:51:43Z

Created page with "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. QL..."

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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 

[[Category:Protocols]]

GuideRNA - Revision history

Paul.xu: Created page with "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-HF3. NEB 44. Ligase5. QL..."

Paul.xu: Created page with "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. QL..."