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

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#Aliquot 5 uL and 10 uL of ligation reactions into eppendorfs
#Aliquot 5 uL and 10 uL of ligation reactions into eppendorfs
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#Pipette 50 uL of competent DH5a
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#Pipette 50 uL of competent DH5a E. coli bacteria into ligation reaction plasmid eppendorfs from above and mix by tapping gently
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#*Do not pipette up and down

Revision as of 09:42, 14 June 2007

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
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