Protocols

Preparing HL5

  • Add approx 2L dH20
  • 88g HL-5 Medium ( HL5 Medium from Formedium cat# HLB0102)
  • 40G Dextrose ( Dextrose Anhydrous, VWR cat# BDH0230-2.5kg)
  • Add stir bar & stir until dissolved
  • Add 2L of dH20 after stirring is completed
  • Pour desired amount into media bottles & autoclave

Preparing SM Agar

  • Obtain ~2800ml glass flask
  • Add 1L dH20
  • Add 41.7g SM Agar (SM Agar from Formedium cat#SMA0102)
  • Add stir bar & stir until dissolved
  • Autoclave
  • Pour desired amount into 3mm dish (~15-18ml)

Preparing SM Plate w/ bacteria

  • Pellet cells down
  • Vac. media,leaving ~200-300ul media
  • Break up pellet
  • Obtain Ka plate, SM Plate, cell scraper, and cell spreader
  • **If using metal cell scraper sterilize using Bunsen burner**
  • Using cell scraper, scrape pff a good pellet of bacteria from KA plate
  • Mix bacterial pellet with broken up cells in tube
  • Pipet up and down to mix & break up any clumps
  • Pipet mixed cells ont the SM plater
  • Obtain cell spreader & sterilize it using Bunsen burner
  • After spreader is cooled, spread cell mixture across plate
  • Leave on bench top for 24hrs, lid side down

Preparing DB Plate w/ cells

  • Microwave DB-Agar (~20sec)
  • Pipet up 3ml DB-Agar & put in 35x10mm petri dish
  • Allow plate to cool
  • Obtain desired cell count (~1.6x10^6 cells/ml)& pellet
  • Resuspend pellet in 1ml DB
  • Pipet up DB/Cells and place onto DB-Agar plate
  • Allow cells to incubate for ~15min
  • Slowly pipet up DB leaving cells firmly attached to DB-Agar
  • Flip plate over & allow to grow

Transformation

  • Pellet cells down (~3ml for 2transformation from 1 confluent plate, 1confluent plate= ~10 trasformations)
  • Wash cells once w/~5ml ice cold H-50 Buffer
  • Resuspend cells in 100ul H-50 buffer
  • Add 1-10ug of DNA (reg use ~5ul)
  • Transfer to a cold 1mm gap eletroporation cuvette
  • Electroporate at .85kV, capacity at 25, Low range to High range, High range set to infinity
  • Slide cuvette in chamber, notch on cuvette faces R
  • Hold down red button until it beeps ( want ~.5 kV reading)
  • Wait 5sec,hold down red button again until it beeps
  • Take out cuvette, & place on ice
  • Transfer cells to 100mm petri dish w/HL5
  • Add appropriate drugs the following day

Preparing H-50 Buffer

  • 20mM Hepes
  • 50mM KCL
  • 10mM NaCL
  • 1mM MgSO4
  • 5mM NaHCO3
  • 1mM NaH2PO4
  • Acquire a PH of 7