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Team:University College London/Protocols
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Latest revision as of 23:39, 18 July 2012
Protocols
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
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.
