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

Grad Advisors

• Morning: Debbie
• 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 uL each)
9 Transformations

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

Transformation Protocol

1. Aliquot 5 uL and 10 uL 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/uL)
2. Pipette 50 uL 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 60 minutes
4. Heat shock the bacteria in a 37C water bath for 30 seconds