image-logoThe Read Lab

Making Bloodstream Form Media

    For 1 L:
  1. Put 500 mL ddH20 into a beaker
  2. Add:
    • 1 pkg of IMDM powder (#12200036 Thermo) = 17.7g
    • 3.025 g Sodium Bicarbonate
    • 184 mg L-cystein
    • 29 mg Bathocuproinedisulfonic acid or 29.9 mg Bathocuproinedisulfonic acid hydrate
  3. Mix 1 hour
  4. Meanwhile - 136 mg hypoxanthine -> 19.8 mL + 200 uL 10 N NaOH -> dissolve on shaker
  5. Add Hypoxanthine
  6. Measure pH - should be really close to 7.4
  7. Bring volume up to 880 mL.
  8. Measure pH - bring to 7.4 if it changed.
  9. Mix 15 min
  10. Filter sterilize
  11. Add:
    • 12.5 mL sodium pyruvate solution
    • 10 mL Penstrep
    • 100 mL FBS
  12. For working stock: add 100 uL of 100 mM B-Me to 100 mL Media
  13. Drug concentrations:
    14. Drug Stock Working conc. uL/100mL media
    G418 100 mg/ml 2 .5 ug/ml 2.5
    Hygromycin B 100 mg/ml 1.5 ug/ml 1.5
    Phleomycin 10 mg/ml 2.5 ug/ml 25
    Puromycin 10 mg/ml 0.1 ug/ml 1
    Blasticidin 10 mg/ml 5 ug/ml 50

Download Protocol: Bloodstream Form Media Protocol
(note requires Microsoft Word to open)