IGEM:MIT/2007/Notebook/2007-7-2
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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
- 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
- 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
- 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)
- Transform FhuA plasmid DNA into E. coli
- Take 10 µg DNA and suspend in 50 µl of water and vortex
- DNA sample now at 200 ng/µL of DNA
- 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
- Put 2 ng of DNA or .8 µL into eppendorf with 50 µL of IAT3 competent E. coli
- Tap gently and incubate on ice for 30 minutes
- Put in 37°C waterbath and heat shock for 20 seconds
- Put in 950 µL of LB+KAN and incubate in warm room shaker for 1 hour
- Decant and resuspend in 900 µL LB and Plate
- Take 10 µg DNA and suspend in 50 µl of water and vortex