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

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