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
 * 2) *Chloramphenicol in freezer at 34 mg/µl (perhaps 1000x)
 * 3) *Guzman used 10 µg/ml CM concentration
 * 4) *For 500 mL LB, need 147 µL of CM
 * 5) *Made 5 labeled tubes filled with 5 mL each of LB and CM
 * 6) Made liquid cultures of E. coli w/ pBAD18 (or 33?)
 * 7) *Picked 5 discrete colonies with loop and innoculated in test tubes incubated in 32°C warm room ovenight
 * 8) Made LB + KAN plates
 * 9) *Take 1.2% LB + Agar and heat in microwave at 50% fro 10 minutes
 * 10) *Wait until cool to ouch and mix in 1000x 950µl of kanamycin
 * 11) *Pour into plates (blue striped)
 * 12) Transform FhuA plasmid DNA into E. coli
 * 13) Take 10 µg DNA and suspend in 50 µl of water and vortex
 * 14) *DNA sample now at 200 ng/µL of DNA
 * 15) Take 1 µL of sample and put in eppendorf with 99 µl of water
 * 16) *DNA sample now at 2 ng/µl
 * 17) *Nanodrop confirms sample at 2.5 ng/µL
 * 18) Put 2 ng of DNA or .8 µL into eppendorf with 50 µL of IAT3 competent E. coli
 * 19) Tap gently and incubate on ice for 30 minutes
 * 20) Put in 37°C waterbath and heat shock for 20 seconds
 * 21) Put in 950 µL of LB+KAN and incubate in warm room shaker for 1 hour
 * 22) Decant and resuspend in 900 µL LB and Plate