diff --git a/wiki/pages/experiments.html b/wiki/pages/experiments.html index af530a60a6e7688a623434af7d7890d4029c0cbb..81e9c32deb84075f6108c8bceb8257588cc8b0f4 100644 --- a/wiki/pages/experiments.html +++ b/wiki/pages/experiments.html @@ -95,15 +95,61 @@ <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 = 1x10ⶠcells/ml)</li> + </ul> + <li>Add 2.5x10⸠cells to 50ml pre-warmed YPD (final concentration: 5x10ⶠcells/ml).</li> + <li>Incubate at 30°C, 200rpm until density reaches 2x10ⷠcells/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;">