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| | <h5>Experiments & Protocols</h5> | | <h5>Experiments & Protocols</h5> |
| | <div style="height:50px"></div> | | <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><br/>
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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>- 1 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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| − | <ul>(if using total volume greater than 10ul, increase the amount of buffer accordingly)</ul><br/>
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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>- 1 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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| − | <ul>(if using total volume greater than 10ul, increase the amount of buffer accordingly)</ul><br/>
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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 <a href="#transformation">transformation protocol</a></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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| − | <br>
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| − | <div id="gel"><h2>Agarose gel electrophoresis</h2>
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| − | <ol>
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| − | <li>Measure 0.50 g of agarose</li>
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| − | <li>Measure 50 ml of 1x TAE buffer using measuring cylinder</li>
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| − | <li> Add agarose and TAE buffer to conical flask and gently mix</li>
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| − | <li>Microwave the flask for 1 min</li>
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| − | <li>Wait for the mixture to cool down slightly before proceeding</li>
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| − | <li>Add 10 ul of 10mg/ml ethidium bromide solution and mix</li>
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| − | <li>Assemble the casting tray and pour the gel into it</li>
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| − | <li>Wait around 30 minutes until gel gets solidified</li>
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| − | <li>Put the gel into the gel chamber and pour 1x TAE buffer until it is fully covered </li>
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| − | <li>Load 6 ul of DNA ladder to the first well.
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| − | <li>Prepare the samples by adding appropriate volume of 6x gel loading dye and load them</li>
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| − | <li>Assemble the gel chamber and run the gel for 40 minutes at 120V</li>
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| − | <li>Visualise the gel using the gel visualizer</li>
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| − | </ol>
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| − | </div>
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| − | <br>
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| − | <div id="gelextraction"><h2>Assembly of 2 parts using gel extraction</h2>
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| − | <ol>
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| − | <li>Digest at least 500 ng of each part according to the <a href="#digestion">restriction digestion</a></li>
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| − | <li>Run the digested DNA on the gel according to <a href="#gel">gel electrophoresis protocol</a></li>
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| − | <li>Identify the parts that you want to ligate on a gel and cut the bands out using razor blade</li>
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| − | <li>Purify the excised bands using the commercial kit according to the manufacturer's instructions</li>
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| − | <li>Quantify the DNA yield using DNA nanodrop</li>
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| − | <li>Proceed to <a href="#ligation">ligation</a></li>
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| − | </ol>
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| − | </div>
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| − | <br>
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| − | <div id="gel"><h2>Polymerase Chain Reaction</h2>
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| − | <ol>
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| − | <li>Prepare the PCR mix:
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| − | <ul>- 12.5 ul of 2 x Q5 PCR master mix</ul>
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| − | <ul>- 1.25 ul of 10 uM forward primer</ul>
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| − | <ul>- 1.25 ul of 10 uM reverse primer</ul>
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| − | <ul>- 2 ng of DNA to be PCRed</ul>
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| − | <ul>- add milliQ H2O up to 25 </ul>
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| − | </li>
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| − | <li> Set up the PCR cycles according to the following rules:
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| − | <ul><b>Initial denaturation</b></ul>
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| − | <ul>- 98C for 30 seconds</ul>
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| − | <ul><b>35 cycles</b></ul>
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| − | <ul>- 98C for 10 seconds</ul>
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| − | <ul>- 30 seconds at primer melting temperature</ul>
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| − | <ul>- 72C for 30sec/kb of PCRed fragment</ul>
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| − | <ul><b>Final extension</b></ul>
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| − | <ul>- 72C for 2 minutes</ul>
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| − | <ul>- Hold at 4C</ul>
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| − | </li>
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| − | <li>- Confirm the PCR by running 2 ul of the product on the gel according to the <a href="#gel">gel electrophoresis protocol</a></li>
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| − | </ol>
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| − | </div>
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| − | <br>
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