Editing CRISPR

== Reagents ==

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

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

<br>3. NEBuffer 4 (New England Biolabs)<br>

4. T4 DNA Ligase (New England Biolabs)<br>

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

6. Kanamycin<br>

7. Chemically competent XL1-Blue cells.<br>

== Protocol ==

<br>1. Digest pDR274 vector (available from Addgene: http://www.addgene.org/crispr/jounglab/CRISPRzebrafish/) using the following conditions:<br>a. 1 µg of pDR274<br>b. 5 µl BsaI-HF<br>c. 5 µl NEB 4<br>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:<br>a. 1 µl 100 µM Oligo 1<br>b. 1 µl 100 µM Oligo 2<br>c. 5 µl 10X annealing buffer<br>d. 43 µl H2O

e. 95 °C<br>f. -1°C per 30 seconds<br>g. 4 °C



5. Ligate annealed oligonucleotides to digested pDR274 vector backbone as follows:<br>a. 1 µl backbone (@ 5ng/µl)<br>b. 3 µl annealed oligo<br>c. 1 µl T4 DNA Ligase<br>d. 5 µl 2X QLB<br>Incubate for 15 minutes at room temperature

<br>6. Transform ligation into XL-1 Blue cells as follows:<br>a. 5 µl of ligation<br>b. 45 µl of chemically competent XL1-Blue cells

c. Ice for 2 min<br>d. 42 ° C for 45 seconds<br>e. Ice for 2 min

f. Add 450 µl of LB and recover at 37°C with shaking for 1 hr<br>g. Plate 100 µl on LB plates containing 30 µg/ml kanamycin <br>h. Grow overnight at 37°C

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

<br>8. Grow cultures overnight at 37°C

<br>9. Isolate plasmids using standard mini-prep procedure

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