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

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 U V (2 uL) dV (5 uL) dV + dI (5 uL) ddV (5 uL) ddV + ddI (5 uL) dV (10 uL) dV + dI (10 uL) ddV (10 uL) ddV + ddI (10 uL)

1. Aliquot 5 uL and 10 uL of ligation reactions into eppendorfs
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