CRISPR

Reagents

1. 10X Annealing Buffer (recipe for making 1 ml):
a. 400 µl 1M Tris @ pH 8
b. 200 µl 1M MgCl2
c. 100 µl 5M NaCl
d. 20 µl 0.5M EDTA @ pH8
e. 280 µl H2O

2. BsaI-HF restriction enzyme (New England Biolabs)

3. NEBuffer 4 (New England Biolabs)

4. T4 DNA Ligase (New England Biolabs)

5. 2X Quick Ligase Buffer (QLB) (New England Biolabs)

6. Kanamycin

7. Chemically competent XL1-Blue cells.

Protocol

1. Digest pDR274 vector (available from Addgene: http://www.addgene.org/crispr/jounglab/CRISPRzebrafish/) using the following conditions:
a. 1 µg of pDR274
b. 5 µl BsaI-HF
c. 5 µl NEB 4
d. H2O to 50 µl

2. Gel isolate digested pDR274 vector backbone

3. Normalize concentration of purified, digested vector to 5 ng/µl in ddH20

4. Anneal two 26 nt oligonucleotides (designed using the web-based ZiFiT Targeter program available here: http://zifit.partners.org) in a thermocycler:
a. 1 µl 100 µM Oligo 1
b. 1 µl 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 annealed oligonucleotides to digested pDR274 vector backbone as follows:
a. 1 µl backbone (@ 5ng/µl)
b. 3 µl annealed oligo
c. 1 µl T4 DNA Ligase
d. 5 µl 2X QLB
Incubate for 15 minutes at room temperature

6. Transform ligation into XL-1 Blue cells as follows:
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 LB plates containing 30 µg/ml kanamycin
h. Grow overnight at 37°C

7. Inoculate single colonies into 1ml of LB supplemented with 30 µg/ml kanamycin

8. Grow cultures overnight at 37°C

9. Isolate plasmids using standard mini-prep procedure

10. Sequence candidate plasmids using M13 primer (see Hwang et al., Nat Biotechnol. 2013 for sequence). Full sequence of plasmid pDR274 is available:  http://www.addgene.org/42250/sequences/.