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

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(Transformation Protocol)
Line 19: Line 19:
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.<br>
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.<br>
-
Start: 2 vials of competent bacteria (500 uL each)<br>
+
Start: 2 vials of competent bacteria (500 µL each)<br>
9 Transformations<br>
9 Transformations<br>
<pre>
<pre>
Contents    Volume    Label
Contents    Volume    Label
-
U V        (2 uL)    I
+
U V        (2 µL)    I
-
dV          (5 uL)    A
+
dV          (5 µL)    A
-
dV + dI    (5 uL)    B
+
dV + dI    (5 µL)    B
-
ddV        (5 uL)    C
+
ddV        (5 µL)    C
-
ddV + ddI  (5 uL)    D
+
ddV + ddI  (5 µL)    D
-
dV          (10 uL)  E
+
dV          (10 µL)  E
-
dV + dI    (10 uL)  F
+
dV + dI    (10 µL)  F
-
ddV        (10 uL)  G
+
ddV        (10 µL)  G
-
ddV + ddI  (10 uL)  H
+
ddV + ddI  (10 µL)  H
</pre><br>
</pre><br>
===Transformation Protocol===
===Transformation Protocol===
-
#Aliquot 5 uL and 10 uL of ligation reactions into eppendorfs
+
#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/uL)
+
#*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)
-
#Pipette 50 uL of competent DH5a E. coli bacteria into ligation reaction plasmid eppendorfs from above and mix by tapping gently
+
#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
#*Do not pipette up and down
#Incubate on ice for 30 minutes
#Incubate on ice for 30 minutes
-
#Heat shock the bacteria in a 37C water bath for 20 seconds
+
#Heat shock the bacteria in a 37°C water bath for 20 seconds
-
#Add 950 uL 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 37C room
+
#Incubate for an hour on a shaker in the 37°C room

Revision as of 10:56, 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
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