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Commit d11e4487 authored by Natalia's avatar Natalia
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<div class="h3">Materials and Reagents</div>
<ul style="text-align:justify; font-family:AccidenzCommons; color:#185A4F; font-weight:400; font-size: min(1.5vw, 22px); font-style: normal; line-height: normal;">
<li>YPD (yeast extract peptone dextrose) liquid media</li>
</ul>
<li>H₂O (sterile)</li>
<li>Polyethylene glycol 3350 (PEG 3350; 50% w/v)</li>
<li>TE buffer (pH 8.0)</li>
<li>Single-stranded carrier DNA (2mg/ml)</li>
<li>1.0M Lithium acetate stock solution (LiAc)</li>
<li>Plasmid DNA and yeast cells</li>
</ul>
<div class="h3">Equipment</div>
<ul style="text-align:justify; font-family:AccidenzCommons; color:#185A4F; font-weight:400; font-size: min(1.5vw, 22px); font-style: normal; line-height: normal;">
<li></li>
</ul>
<li>Spectrophotometer</li>
<li>Water baths (30°C, 42°C, 96°C)</li>
<li>Shaking incubator</li>
<li>Centrifuge</li>
<li>Vortex mixer</li>
<li>Micropipettes and sterile tips</li>
<li>Microfuge tubes and 50ml centrifuge tubes</li>
</ul>
<div class="h3">Procedure</div>
<ol style="text-align:justify; font-family:AccidenzCommons; color:#185A4F; font-weight:400; font-size: min(1.5vw, 22px); font-style: normal; line-height: normal;">
<li></li>
</ol>
<li>Inoculate yeast cells in 100ml YPD and grow overnight at 30°C, 200rpm.</li>
<li>After 12-16h, measure culture density:</li>
<ul>
<li>Dilute 10μl cells in 1ml water</li>
<li>Measure OD₆₆₀ (0.1 OD₆₆₀ = 1x10⁶ cells/ml)</li>
</ul>
<li>Add 2.5x10⁸ cells to 50ml pre-warmed YPD (final concentration: 5x10⁶ cells/ml).</li>
<li>Incubate at 30°C, 200rpm until density reaches 2x10⁷ cells/ml (~4 hours).</li>
<li>Harvest cells: centrifuge at 3000g for 5 minutes.</li>
<li>Wash cells with 25ml sterile H₂O, and centrifuge again.</li>
<li>Resuspend cell pellet in 1.0ml of 0.1M LiAc, transfer to 1.5ml microfuge tube.</li>
<li>Centrifuge at top speed for 5 seconds, and remove LiAc.</li>
<li>Resuspend cells in 0.1M LiAc to a final volume of 500μl (about 400μl LiAc).</li>
<li>Prepare carrier DNA: boil 1.0ml sample for 5 minutes, then chill on ice.</li>
<li>Aliquot 50μl of cell suspension into labeled microfuge tubes, centrifuge, and remove LiAc.</li>
<li>Add transformation mix in this order:</li>
<ul>
<li>240μl PEG 3350 (50% w/v)</li>
<li>36μl 1.0M LiAc</li>
<li>25μl single-stranded carrier DNA (2.0mg/ml)</li>
<li>50μl H₂O + plasmid DNA (0.1-10μg)</li>
</ul>
<li>Vortex vigorously until the cell pellet is completely mixed (~1 minute).</li>
<li>Incubate 30 minutes at 30°C.</li>
<li>Heat shock for 30 minutes at 42°C.</li>
<li>Centrifuge at 6000-8000rpm for 15s, remove transformation mix.</li>
<li>Resuspend pellet in 0.2ml sterile H₂O.</li>
<li>Plate cells in different concentrations on selective plates. Recommended:</li>
<ul>
<li>10μl cells + 100μl H₂O</li>
<li>200μl cells undiluted</li>
</ul>
<li>Incubate plates at 30°C for 3 days.</li>
</ol>
<div class="h2"><i>C. reinhardtii </i> Transformation</div>
<div class="h3">Materials and Reagents</div>
<ul style="text-align:justify; font-family:AccidenzCommons; color:#185A4F; font-weight:400; font-size: min(1.5vw, 22px); font-style: normal; line-height: normal;">
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