IGEM:MIT/2007/Notebook/2007-7-2

From OpenWetWare

Jump to: navigation, search

Agenda: Monday, 7/2

  • Order stuff!
  • Summary email to all iGEM


pBAD33

  • Received pBAD33 promoter and regulatory protein inside pBAD18/24 vector with chloramphenicol resistance
    • Maybe actually the pBAD33 vector
  • Need to make liquid culture inside LB and CMR
  1. Make LB and CM
    • Chloramphenicol in freezer at 34 mg/µl (perhaps 1000x)
    • Guzman used 10 µg/ml CM concentration
    • For 500 mL LB, need 147 µL of CM
    • Made 5 labeled tubes filled with 5 mL each of LB and CM
  2. Made liquid cultures of E. coli w/ pBAD18 (or 33?)
    • Picked 5 discrete colonies with loop and innoculated in test tubes incubated in 32°C warm room ovenight
  3. Made LB + KAN plates
    • Take 1.2% LB + Agar and heat in microwave at 50% fro 10 minutes
    • Wait until cool to ouch and mix in 1000x 950µl of kanamycin
    • Pour into plates (blue striped)
  4. Transform FhuA plasmid DNA into E. coli
    1. Take 10 µg DNA and suspend in 50 µl of water and vortex
      • DNA sample now at 200 ng/µL of DNA
    2. Take 1 µL of sample and put in eppendorf with 99 µl of water
      • DNA sample now at 2 ng/µl
      • Nanodrop confirms sample at 2.5 ng/µL
    3. Put 2 ng of DNA or .8 µL into eppendorf with 50 µL of IAT3 competent E. coli
    4. Tap gently and incubate on ice for 30 minutes
    5. Put in 37°C waterbath and heat shock for 20 seconds
    6. Put in 950 µL of LB+KAN and incubate in warm room shaker for 1 hour
    7. Decant and resuspend in 900 µL LB and Plate
Personal tools