IGEM:MIT/2007/Notebook/2007-6-14

Grad Advisors

• Morning: Debbie, Forrest
• Afternoon: Brian

LAB WORK

• Make competent E. coli cells
• Transformation into competent cells
• Plate transformed cells

Depending on how fast cells grow......

• Night time
  • Analyze plates
  • innoculate 4ml LB/Amp

Procedures

Details of DH5 Strain E Coli

Regular cell membranes are not permeable to plasmids, so we much use ions (CaCl) to make the bacteria competent and able to accept foreign DNA.
Start: 2 vials of competent bacteria (500 µL each)
9 Transformations

Contents    Volume    Label
U V         (2 µL)    I
dV          (5 µL)    A
dV + dI     (5 µL)    B
ddV         (5 µL)    C
ddV + ddI   (5 µL)    D
dV          (10 µL)   E
dV + dI     (10 µL)   F
ddV         (10 µL)   G
ddV + ddI   (10 µL)   H

Transformation Protocol

1. Aliquot 5 µL and 10 µL of ligation reactions into eppendorfs
   • For the undigested vector, dilute 100x so we can put in around 2 ng (because optical density of undigested vector is 86.4 ng/µL)
2. Pipette 50 µL of competent DH5a E. coli bacteria into ligation reaction plasmid eppendorfs from above and mix by tapping gently
   • Do not pipette up and down
3. Incubate on ice for 30 minutes
4. Heat shock the bacteria in a 37°C water bath for 20 seconds
5. Add 950 µL LB to each of the eppendorf tubes
6. Incubate for an hour on a shaker in the 37°C room
7. Centrifuge at 8000 rpm for 5 minutes
8. Decant 900 uL of supernatant and resuspend the rest of the cells
9. Take the contents of tubes and spread them on LB Amp plates
   • B has a hole, G is barbequed toast, literally

Project Research Homework

• Aditya: sensory promotors / metal ion binding
• Alex, Bernice: past iGEM projects (2006, 2005)
• Semmie,Jessica: Hwang papers (mussel proteins)
• Toan: Rice07 (read for bacterial display)