Team:University College London/Protocols

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<html><div id="EnzDig" class="protocol" style="background-image:url('https://static.igem.org/mediawiki/2012/d/de/Ucl2012-protocols-logo-enzdig.png')">Enzyme Digest</div></html><div class="protocolContent">  
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<html><div id="EnzDig" class="protocol" style="background-image:url('https://static.igem.org/mediawiki/2012/d/de/Ucl2012-protocols-logo-enzdig.png')">Enzyme Digest</div><div class="protocolContent"></html>{{:Team:University_College_London/Protocols/EnzDig}}<html></div>
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1. Thaw all materials on ice
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<div id="Transformation" class="protocol" style="background-image:url('https://static.igem.org/mediawiki/2012/f/fd/Ucl2012-protocols-logo-transformation.png')">Transformation</div>
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2. Add the following ingredients to autoclaved/sterile eppendorf tubes
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Insert Table
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3.Flick contents gently and centrifuge.
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4.To centrifuge, add sample eppendorf + balancer (fill another tube with equal volume of water if only have one sample) and screw lid on tight
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5. Then run on the following conditions (by turning the dial)
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RPM: 14000
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Time: 1 minute
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Temperature: 18oC
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<html><div id="Transformation" class="protocol" style="background-image:url('https://static.igem.org/mediawiki/2012/f/fd/Ucl2012-protocols-logo-transformation.png')">Transformation</div></html>
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1.Thaw competent cells on ice.
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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.
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4.Close tube and incubate the cells on ice for 30 minutes.
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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.
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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. 
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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.
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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.
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11.You can pick a single colony, make a glycerol stock, grow up a cell culture and miniprep.
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Latest revision as of 23:39, 18 July 2012

Protocols

Enzyme Digest

Step 1 - Thawing cells: Thaw all materials on ice

Step 2 - Adding Ingredient: Add the following ingredients to autoclaved/sterile eppendorf tubes

Component Amount (ul) (one enzyme used) Amount (ul) (two enzymes used)
dH20 2.5 1.5
Buffer 1x 1 1
DNA template 5 5
BSA 0.5 0.5
Enzyme 1 1 2
Enzyme 2 N/A 1


Step 3 - Addition of BioBrick: Flick contents gently and centrifuge.

Step 4 - Centrifuge:

RPM: 14000

Time: 1 minute

Temperature: 18oC

Step 5 - Digest Program: Place the samples on a thermocycler under the following conditions:

RPM: 550

Time: 2.5 hours

Temperature: 37oC

Step 6 - Denaturing Enzymes: If you are not running the samples on a gel immediately, denature the restriction enzymes by running the samples on a thermocycler under the following conditions:

RPM: 550

Time: 25 minutes

Temperature: 65oC


Transformation

Step 1 - Thawing Cells: Thaw competent cells on ice.

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

Step 3 - Addition of BioBrick: 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.

Step 4 - Incubation: Close tube and incubate the cells on ice for 30 minutes.

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

Step 6 - Incubation: Incubate the cells on ice for 5 minutes.

Step 7 - Add media: Add 200 μl of SOC media or LB broth

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

Step 9 - Label plates: 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.

Step 10 - Culture: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.

Step 11 - Colony Picking: You can pick a single colony, make a glycerol stock, grow up a cell culture and miniprep.


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