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

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(Transformation Protocol)
Line 44: Line 44:
#Add 950 µL LB to each of the eppendorf tubes
#Add 950 µL LB to each of the eppendorf tubes
#Incubate for an hour on a shaker in the 37°C room
#Incubate for an hour on a shaker in the 37°C room
 +
#Centrifuge at 8000 rpm for 5 minutes
 +
#Decant 900 uL of supernatant and resuspend the rest of the cells
 +
#Take the contents of tubes and spread them on LB Amp plates

Revision as of 12:03, 14 June 2007

Contents

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 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
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