Team:University College London/Protocols/Transformation2

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<div class="protocol-transformation"> == Transformation == </div>
<div class="protocol-transformation"> == Transformation == </div>
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1.Thaw competent cells on ice.
1.Thaw competent cells on ice.
 +
2.Add 50 µL of thawed competent cells into pre-chilled 2ml tube.
2.Add 50 µL of thawed competent cells into pre-chilled 2ml tube.
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3.Add 1 - 2 µL of the resuspended DNA to the 2ml tube. Pipette up and down a few times, gently. Make sure to keep the competent cells on ice.
3.Add 1 - 2 µL of the resuspended DNA to the 2ml tube. Pipette up and down a few times, gently. Make sure to keep the competent cells on ice.
 +
4.Close tube and incubate the cells on ice for 30 minutes.
4.Close tube and incubate the cells on ice for 30 minutes.
 +
5.Heat shock the cells by immersion in a pre-heated water bath at 42ºC for 60 seconds.
5.Heat shock the cells by immersion in a pre-heated water bath at 42ºC for 60 seconds.
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6.Incubate the cells on ice for 5 minutes.
6.Incubate the cells on ice for 5 minutes.
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7.Add 200 μl of SOC media or LB broth
7.Add 200 μl of SOC media or LB broth
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8.Incubate the cells at 37ºC for 2 hours while the tubes are rotating or shaking. 
8.Incubate the cells at 37ºC for 2 hours while the tubes are rotating or shaking. 
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9.Label two petri dishes with LB agar and the appropriate antibiotic(s) with the part number, plasmid backbone, and antibiotic resistance. Plate 20 µl and 200 µl of the transformation onto the dishes, and spread. This helps ensure that you will be able to pick out a single colony.
9.Label two petri dishes with LB agar and the appropriate antibiotic(s) with the part number, plasmid backbone, and antibiotic resistance. Plate 20 µl and 200 µl of the transformation onto the dishes, and spread. This helps ensure that you will be able to pick out a single colony.
 +
10.Incubate the plate at 37ºC for 12-14 hours, making sure the agar side of the plate is up. If incubated for too long the antibiotics start to break down and un-transformed cells will begin to grow. This is especially true for ampicillin - because the resistance enzyme is excreted by the bacteria, and inactivates the antibiotic outside of the bacteria.
10.Incubate the plate at 37ºC for 12-14 hours, making sure the agar side of the plate is up. If incubated for too long the antibiotics start to break down and un-transformed cells will begin to grow. This is especially true for ampicillin - because the resistance enzyme is excreted by the bacteria, and inactivates the antibiotic outside of the bacteria.
 +
11.You can pick a single colony, make a glycerol stock, grow up a cell culture and miniprep.
11.You can pick a single colony, make a glycerol stock, grow up a cell culture and miniprep.

Revision as of 15:28, 16 July 2012

== Transformation ==


1.Thaw competent cells on ice.

2.Add 50 µL of thawed competent cells into pre-chilled 2ml tube.

3.Add 1 - 2 µL of the resuspended DNA to the 2ml tube. Pipette up and down a few times, gently. Make sure to keep the competent cells on ice.

4.Close tube and incubate the cells on ice for 30 minutes.

5.Heat shock the cells by immersion in a pre-heated water bath at 42ºC for 60 seconds.

6.Incubate the cells on ice for 5 minutes.

7.Add 200 μl of SOC media or LB broth

8.Incubate the cells at 37ºC for 2 hours while the tubes are rotating or shaking. 

9.Label two petri dishes with LB agar and the appropriate antibiotic(s) with the part number, plasmid backbone, and antibiotic resistance. Plate 20 µl and 200 µl of the transformation onto the dishes, and spread. This helps ensure that you will be able to pick out a single colony.

10.Incubate the plate at 37ºC for 12-14 hours, making sure the agar side of the plate is up. If incubated for too long the antibiotics start to break down and un-transformed cells will begin to grow. This is especially true for ampicillin - because the resistance enzyme is excreted by the bacteria, and inactivates the antibiotic outside of the bacteria.

11.You can pick a single colony, make a glycerol stock, grow up a cell culture and miniprep.

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