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

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(Procedures)
(Transformation Protocol)
Line 40: Line 40:
#Pipette 50 uL of competent DH5a E. coli bacteria into ligation reaction plasmid eppendorfs from above and mix by tapping gently
#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
#*Do not pipette up and down
-
#Incubate on ice for 60 minutes
+
#Incubate on ice for 30 minutes
-
#Heat shock the bacteria in a 37C water bath for 30 seconds
+
#Heat shock the bacteria in a 37C water bath for 20 seconds
 +
#Add 950 uL LB to each of the eppendorf tubes
 +
#Incubate for an hour on a shaker

Revision as of 09:33, 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 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 30 minutes
  4. Heat shock the bacteria in a 37C water bath for 20 seconds
  5. Add 950 uL LB to each of the eppendorf tubes
  6. Incubate for an hour on a shaker
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