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Team:TU-Delft/Protocol 1
From 2013.igem.org
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<h4 align="left">Requirements:</h4> | <h4 align="left">Requirements:</h4> | ||
| - | + | <ol> | |
| - | + | <li> Distilled water</li> | |
| - | + | <li>Pipettes</li> | |
| - | + | <li> Ice </li> | |
| - | + | <li> Competent cells</li> | |
| - | + | <li> SOC media</li> | |
| - | + | <li> Agar plates with Antibiotics</li> | |
| - | + | <li> Timer</li> | |
| + | <li> Water bath</li> | ||
| + | </ol> | ||
<br> | <br> | ||
<h2 align="center">Prodecure</h2> | <h2 align="center">Prodecure</h2> | ||
| - | + | <ol> | |
| - | + | <li> Mark the location on distribution plate by looking at the right row and column as mentioned. Punch the plate with a pipette and add 10 µL of distilled water. </li> | |
| - | + | <li> Resuspend the Dry DNA on the distribution plate with distilled water. </li> | |
| - | + | <li> Inoculate the competent cells with the desired DNA from the distribution plate as described in step 2. Add 1 µL of the resuspended DNA to the competent cells. Use 2 mL tubes and keep the cells on ice at all the times for a more efficient transformation. </li> | |
| - | + | <li> Incubate the transformation on ice for 5 min. </li> | |
| - | + | <li> Heat shock the transformation the water bath at 42⁰C for 30 sec.</li> | |
| - | + | <li> Incubate on ice for 2 min. </li> | |
| - | + | <li> Add 200 µL SOC Media to the transformation.</li> | |
| - | + | <li> Incubate the transformation at 37⁰C for 1 hour. </li> | |
| - | + | <li> Plate out 20 µL of transformation with 10% and 90% on separate agar plates with appropriate antibiotics.</li> | |
| - | + | <li> Incubate the agar plate overnight (14-16 hours) at 37⁰C.</li> | |
| + | </ol> | ||
<br> | <br> | ||
| - | |||
Single colonies are selected from the above plates and grown in LB media to prepare glycerol stocks and carry out Miniprep to extract the desired plasmid. | Single colonies are selected from the above plates and grown in LB media to prepare glycerol stocks and carry out Miniprep to extract the desired plasmid. | ||
</p> | </p> | ||
</html> | </html> | ||
Revision as of 12:17, 29 September 2013
Protocols
Our project deals with E.coli cells which sense Auto-inducing peptides (AIPs) from the Staphylococcus aureus and starts producing Antimicrobial peptides in order to kill the Staphylococcus aureus. Different protocols used during the project are described below.
- Transforming Parts from Distribution kit
- Growing the Single Colonies from the Agar Plates
- Making glycerol stocks
- Miniprep Protocol
- Restriction digestion
- Ligation
- Gel Extraction Procedure
- PCR Purification
Transforming Parts from Distribution kit
Requirements:
- Distilled water
- Pipettes
- Ice
- Competent cells
- SOC media
- Agar plates with Antibiotics
- Timer
- Water bath
Prodecure
- Mark the location on distribution plate by looking at the right row and column as mentioned. Punch the plate with a pipette and add 10 µL of distilled water.
- Resuspend the Dry DNA on the distribution plate with distilled water.
- Inoculate the competent cells with the desired DNA from the distribution plate as described in step 2. Add 1 µL of the resuspended DNA to the competent cells. Use 2 mL tubes and keep the cells on ice at all the times for a more efficient transformation.
- Incubate the transformation on ice for 5 min.
- Heat shock the transformation the water bath at 42⁰C for 30 sec.
- Incubate on ice for 2 min.
- Add 200 µL SOC Media to the transformation.
- Incubate the transformation at 37⁰C for 1 hour.
- Plate out 20 µL of transformation with 10% and 90% on separate agar plates with appropriate antibiotics.
- Incubate the agar plate overnight (14-16 hours) at 37⁰C.
Single colonies are selected from the above plates and grown in LB media to prepare glycerol stocks and carry out Miniprep to extract the desired plasmid.
