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| − | <h5>Experiments & Protocols</h5>
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| − | <div style="height:50px"></div>
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| − | <p>Describe the experiments, research and protocols you used in your iGEM project.</p>
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| − | <ul>
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| − | <li> Protocols </li>
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| − | <li> Experiments </li>
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| − | <li>Documentation of the development of your project </li>
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| − | </ul>
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| − | <div id="digestion"><h2>Restriction digestion protocol</h2>
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| − | <ol>
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| − | <li>Prepare restriction digestion mixture:
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| − | <ul><b>IDT gBlocks</b></ul>
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| − | <ul>- 10 ul of DNA (10 ng/ul)</ul>
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| − | <ul>- 2 ul of 10 x 2.1 buffer</ul>
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| − | <ul>- 0.3 ul of EcoR1</ul>
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| − | <ul>- 0.3 ul of Pst1</ul>
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| − | <ul>- 7.4 ul of milliQ H2O</ul>
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| − | <ul><b>PSB1C3, PSB1A3, PSB1K3 backbones</b></ul>
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| − | <ul>- Required amount of vector DNA (50 ng per ligation) </ul>
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| − | <ul>- 2 ul of 10 x 2.1 buffer</ul>
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| − | <ul>- 0.3 ul of EcoR1</ul>
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| − | <ul>- 0.3 ul of Pst1</ul>
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| − | <ul>- add milliQ H2O up to 10 ul</ul>
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| − |
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| − | <ul><b>Other digestions</b></ul>
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| − | <ul>- Required amount of vector DNA</ul>
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| − | <ul>- 2 ul of 10 x 2.1 buffer</ul>
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| − | <ul>- 0.3 ul of EcoR1</ul>
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| − | <ul>- 0.3 ul of Pst1</ul>
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| − | <ul>- add milliQ H2O up to 10 ul</ul>
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| − | </li>
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| − | <li>Incubate at 37C for an hour</li>
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| − | <li> Heat inactivate by incubating at 80C for 20 minutes</li>
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| − | <li>Run a sample of digested DNA on a gel in order to confirm digestion:
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| − | <ul>- 2 ul of DNA</ul>
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| − | <ul>- 1 ul of 6 x Gel Loading Dye</ul>
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| − | <ul>- 3 ul of milliQ H2O</ul>
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| − | </li>
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| − | <li>If digestion is confirmed, proceed to ligation</li>
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| − | </ol>
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| − | </div>
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| − | <br>
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| − | <div id="ligation"><h2>Ligation protocol</h2>
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| − | <ol>
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| − | <li> Calculate the amount of insert DNA required to maintain 1:3 backbone:insert molar ratio using formula below. For standard ligations use 50 ng of vector DNA, increase the amounts of DNA if unsuccessful.<br/>
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| − | <img src="https://static.igem.org/mediawiki/2015/9/9a/UCL_ligationformula.png.png">. </li>
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| − | <li> Prepare the ligation mixture:
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| − | <ul> - required amount of insert DNA</ul>
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| − | <ul> - required amount of vector DNA </ul>
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| − | <ul> - 1 ul of T4 ligase </ul>
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| − | <ul> - 2 ul of 10 x ligase buffer </ul>
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| − | <ul> - add milliQ H2O up to 20 ul</ul>
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| − | </li>
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| − | <li>Incubate at 16C for 30 minutes</li>
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| − | <li>Heat inactivate by incubating at 80C for 20 minutes</li>
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| − | <li>Keep on ice until ready to proceed with transformation protocol</li>
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| − | </ol>
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| − | </div>
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| − | <br/>
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| − | <div id="transformation"><h2>Transformation protocol </h2>
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| − | <ol>
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| − | <li>(If using part from the distribution: resuspend the DNA in 10 ul of MiliQ water, making sure that it turns red. Wait 10 minutes before adding the DNA to cells)</li>
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| − | <li>Put a tube of NEB DH 5 alpha <i>E. coli</i> cells on ice and wait until they thaw completely. Divide the cells into 50 ul aliquotes. </li>
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| − | <li>Add 1 ul of plasmid DNA to 50 ul of cells. </li>
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| − | <li>Mix by carefully flicking the tube. Do not vortex or pipette in and out! </li>
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| − | <li>Place the mixture on ice for 30 minutes. </li>
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| − | <li>Heat shock the cells at 42 °C for 30 seconds and immediately put on back on ice.</li>
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| − | <li>Keep cells on ice for next 5 minutes. Do not mix. </li>
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| − | <li>Pipette 950 ul of SOC media kept at room temperature into the mixture. If SOC is not available, use LB. <br/>
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| − | <li>Incubate the mixture at 37 °C for 60 minutes </li>
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| − | <li>Prepare plates with appropriate antibiotics. Bring plates to room temperature before plating. Use 2 plates per transformation reaction. </li>
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| − | <li>Plate 200 ul of cells on one plate.
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| − | <li>Pellet the remaining cells and resuspend in 200ul of LB.</li>
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| − | <li> Plate the remaining cells on second plate.</li>
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| − | <li>Incubate plates overnight at 37 °C. </li>
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| − | </ol>
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| − | </div>
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