Protocols

Preparing Agar Plates Protocol (Makes 12 (15mm) Plates)

• 300 mL DI H2O + 11 g LB agar
• Autoclave
  • Put control thermometer in H₂O (from the sink)
  • Select vented container mode (Do Not Change Program)
• Mix well after autoclaving; let cool to 50^oC
• Add antibiotic (50 to 100 µg/mL) (15 mg total)
  • Weigh on paper
  • Add to 0.5 mL DI H₂O
  • Add to LB mixture when cool enough
• Plate
  • Under flame open lids of all plates
  • Slowly pour agar into plate, avoiding bubbles, when it touches all edges stop pouring
  • Let sit under flames until gel solidifies
  • Replace lids on plates
• Store upside down at 40^oC

Preparing Competent Cells Protocol

• Place 1 colony in 5 mL of LB (with antibiotics if appropriate) Grow overnight at 37^oC and 200-300 rpm
• Inoculate 0.25 mL of the overnight strain into 25 mL of LB
• Shake at 37^oC until the OD650 is 0.6-0.7
• Harvest cells and resuspend in 12.5 mL ice cold 0.1M MgCl₂
• Harvest immediately and resuspend in 7.5 mL cold 0.1M CaCl₂
• Leave on ice for 30 minutes. Harvest and resuspend in 2.5 mL cold 0.1M CaCl₂
• Leave on ice for 30 minutes
• For long term storage, use 0.1M CaCl₂ in 15% glycerol at step 6 and store cells at -800^oC

Note: Harvest cells at 5000 rpm for 10 minutes at 40^oC