Sinton:Protocols/Ferric BG-11 Media preparation

From OpenWetWare

Jump to: navigation, search

Contents

Safety

  • Safety goggles
  • Nitrile gloves
  • Lab coat
  • Biosafety cabinet

Consumeables

  • Weigh boat
  • Autoclave tape
  • Syringe
  • Needle
  • Pipette (4)

Equipment

  • Clean 1 L bottle
  • 10 100mL bottles
  • Scoopula or similar device
  • Precision Mass Balance

Chemicals needed

  • Stock Solution I,II, IV, V
  • Ferric ammonium Citrate (check bottle)
  • Citric Acid (check bottle)

Protocol

  • Clean all Glassware!!
  • Fill 1L bottle to 500-750mL with DI water
  • Add the Following stock solutions to the DI water in the 1 L bottle
     *10mL Stock I 
     *10mL Stock II
     *40mL Stock IV
     *1mL Stock V
  • Add 6mg of Ferric ammonium Citrate
  • Add 6mg of Citric Acid
     *THESE STEPS ARE 'difficult' due to the small quantities if you can weight it out well done
     OR
     *Weigh out 30mg in a weight boat using a precision mass balance
     *Add 5mL of of Water to weigh boat, remove 1mL and add to 1L bottle
     NOTE: This would be Stock III anecdotally, this solution was very susceptible to contamination due to Vitamin C presence
  • Top up Media bottle (1L) to 1L
  • SHAKE IT don't BREAK IT
  • Dispense Media into 100mL bottles, filling precisely to either 100,80 or 60, this is to enable easier addition of any antibiotic agents
  • Autoclave bottles
  • Once cooled, transfer bottles from autoclave to biosafety cabinet
  USE CAUTION IN THE NEXT STEP (SHARPS)
  • Extract 6mL of Kanamycin (10mg/mL) and tranfer to weighboat or test tube etc...
  • using a pipette add 5microliters/mL to each bottle
  • Seal bottles and remove from biosafety cabinet
  • label bottle
   *Date
   *FBG11
   *Kanamycin concentration used (5uL/mL)
Personal tools