Equipment and Materials

Week 1

• UV-Vis Spectrophotometer for DNA quantification and bacterial cell density measurements
• MicroPulser Electroporator (BioRad USA, Hercules, CA)
• Sterile 1 mm Gene Pulser cuvette (BioRad USA, Hercules, CA)
• Refrigerated centrifuge with a Sorvall SA-600 rotor set at 4°C (Thermo Fisher Scientific, Wilmington, DE)
• Prechilled 50 ml sterile Oakridge tube
• Refrigerated Microcentrifuge set at 4°C
• Ice chest
• Shaking water bath set at 42°C
• Incubator set at 30–32°C
• 40 ml LB broth in 250 ml flask
• 50 ml sterile ice-cold water
• Micropipettes and filter pipette tips (20 µl, 200 µl, and 1000 µl)
• 1.5 ml prechilled microcentrifuge tube
• 15 ml Falcon/culture tube (17 mm × 100 mm)
• Bacterial spreader
• Parafilm strips
• Recombineering competent cells: E. coli strain HME 63 (Grow at 30–32°C)
• Single-stranded oligodeoxyribonucleotide (ssODN, sense strand) (100 pmol per group) Yellow-highlight/bold is the PAM sequence with two-nucleotide substitution underlined
  • 5′-ATGTACTCGTGTATATACTACCACTCCTACCAAACCGAACTCCGCGCTGCGTAAAGTAT-3′
• anti-ssODN (antisense strand) (100 pmol per group)
  • 5′-ATACTTTACGCAGCGCGGAGTTCGGTTTGGTAGGAGTGGTAGTATATACACGAGTACAT-3′
• LB agar plates (3 each per group)
  • LB agar
  • LB agar containing streptomycin (50 µg/ml)

Weeks 2 and 4

• Bio-Rad T100 PCR Thermal Cycler or similar (BioRad USA, Hercules, CA)
• Agarose gel electrophoresis apparatus (BioRad USA, Hercules, CA)
• Bio-Rad GelDoc Go Imaging System (BioRad USA, Hercules, CA) or similar
• NanoDrop 1000 (Thermo Fisher Scientific, Wilmington, DE)
• Microcentrifuge speed ≥ 13,000 rpm
• Shaking water bath (42°C), shakers (30–32°C), incubator (30–32°C)
• Ice chest
• 0.2 ml PCR tubes
• Micropipette, pipette tips, and filter pipette tips (20 µl, 200 µl, and 1000 µl)
• Ice buckets
• ThermoScientific Phusion™ DNA polymerase (2U/µl) with proofreading (Thermo Fisher Scientific, Wilmington, DE)
• GeneJET™ PCR Purification kit (Thermo Fisher Scientific, Wilmington, DE)
• 50X TAE running buffer
• GelRed DNA binding dye (Biotium, Inc. Fremont, CA)
• 6X DNA loading buffer
• DNA molecular weight marker (100 bp ladder)
• 3 M sodium acetate, pH 5.2
• 1.5 ml microcentrifuge tubes

Week 3

• UV-Vis Spectrophotometer for DNA quantification and bacterial cell density measurements
• MicroPulser Electroporator (BioRad USA, Hercules, CA)
• Sterile 1 mm Gene Pulser cuvette (BioRad USA, Hercules, CA)
• Refrigerated centrifuge with a Sorvall SA-600 rotor set at 4°C (Thermo Fisher Scientific, Wilmington, DE)
• 50 ml prechilled sterile Oakridge tube
• 15 ml Falcon/culture tube (17 mm × 100 mm)
• 1.5 ml microfuge tube
• Bacterial spreader
• Parafilm strips for sealing the plates
• The following bacterial strains are gifts from Luciano Marraffini.
  • Bacterial strain containing Cas9 (Addgene plasmid # ­42876; RRID:­Addgene_­42876)
  • Bacterial strain containing pCRISPR::Φ (Addgene ­plas­mid ­# ­42875; RRID:­Addgene_42875)
  • Bacterial strain containing pCRISPR::rpsL (Addgene ­plas­mid # ­44505; RRID:­Addgene_­44505)
• 50 ml sterile ice-cold water (one per two groups).
• Recombineering proficient E. coli strain HME63 containing pCas9-tracrRNA (Grown in LB containing 25 µg/ml chloramphenicol at 32°C)
• pCRISPR::rpsL (300 ng per reaction)
• pCRISPR::Φ (300 ng per reaction)
• antisense-ssODN (100 pmol per reaction)
• 40 ml LB broth in 250 ml flask (one per two groups)
• LB agar plates (2 each, per group)
  • LB agar
  • LB kan Agar containing 25 µg/ml kanamycin
  • LB strep Agar containing 50 µg/ml streptomycin
  • LB kan-strep Agar containing 25 µg/ml kanamycin and 50 µg/ml streptomycin

**Preparing the electroporation cuvettes

1. Wash cuvettes with H₂O, rinse with sterile RO H₂O, and dry
2. Store them at 75% EtOH
3. Before electroporation, dry them again