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- | <html> | + | <center> |
| + | =Protocols - Overview= |
| + | </center> |
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- | | + | In this section we are going to note all protocols that the teams have used for their work. |
- | | + | <center> |
- | <h1 align=center> This page lists all molecular genetics protocols we use in our project </h1>
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- | | + | | [https://2012.igem.org/Team:Bielefeld-Germany/Protocols/Genetics General Protocols] || Team Cloning || Team Cultivation |
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- | = Complete genome isolation from yeast with the Promega Wizard genomic DNA purification system kit =
| + | | [https://2012.igem.org/Team:Bielefeld-Germany/Protocols/TeamActivity Team Activity Test] || Team Immobilization || Team Substrate |
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| + | | Team Yeast || Team Sequencing || Team Modelling |
- | <p align=justify><font>
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- | | + | |} |
- | <ul>
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- | <li>Pellet 10 mL of over-night liquid culture grown in YPD broth in a 1.5 mL tube by centrifugation at 14,000 x g for 2 minutes.</li>
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- | <li>Remove the supernatant.</li>
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- | <li>Resuspend the cells in 90 μL of 50 mM EDTA.</li>
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- | <li>Add 10 μL of 1000u lyticase and pipet 4 times to mix.</li>
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- | <li>Incubate the sample at 37°C for 60 minutes to digest the cell wall</li>
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- | <li>Centrifuge the sample at 14,000 × g for 2 minutes and then remove the supernatant.</li>
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- | <li>Add 300 μl of Nuclei Lysis Solution to the cell pellet and pipet to mix.</li>
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- | <li>Add 100 μl of Protein Precipitation Solution and vortex at high speed for 20 seconds.</li>
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- | <li>Let the sample sit on ice for 5 minutes.</li>
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- | <li>Centrifuge at 14,000 × g for 3 minutes.</li>
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- | <li>Transfer the supernatant containing the DNA to a clean 1.5 ml tube containing 300 μl of room temperature isopropanol.</li>
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- | <li>Gently mix by inversion until the DNA is visible.</li>
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- | <li>Centrifuge at 14,000 × g for 2 minutes.</li>
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- | <li>Carefully decant the supernatant and drain the tube on clean absorbent paper.</li>
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- | <li>Add 300 μl of room temperature 70% ethanol and invert the tube several times to wash the DNA pellet.</li>
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- | <li>Centrifuge at 14,000 × g for 2 minutes.</li>
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- | <li>Drain the tube on clean absorbent paper and allow the pellet to air-dry for 15 minutes.</li>
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- | <li>Add 50 μl of DNA Rehydration Solution.</li>
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- | <li>Add 1.5μl of RNase Solution to the purified DNA sample. Vortex the sample for 1 second and incubate at 37°C for 15 minutes.</li>
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- | <li>Rehydrate the DNA by incubating at 65°C for 1 hour. Periodically mix the solution by gently tapping the tube.</li>
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- | <li>Store the DNA at 2–8°C.</li>
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- | </ul>
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- | </font></p>
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- | <h2 align=left><i>Arabidopsis thaliana</i>: Growth Conditions and Plant Material</h2>
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- | <p align=justify><font>
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- | Six weeks old <i>A. thaliana</i> plants, ecotype Columbia 0 (wildtype), have been gratefully offered by Patrick Treffon and Thorsten Seidel. They have been cultivated under normal day conditions (14 hours light [100 µmol ⁄ quanta m<sup>-2</sup>s<sup>-1</sup>] at 21°C, 10 hours darkness at 18°C). For induction of the formation of siliques the plants were shifted into long day conditions (16 hours light [100 µmol ⁄ quanta m<sup>-2</sup>s<sup>-1</sup>] at 21°C, 18 hours darkness at 18°C). After two weeks in long day conditions the plants have developed 2 cm long siliques. The siliques were harvested and frozen in liquid nitrogen for further use.
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- | </font></p>
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- | <h2 align=left><i>Arabidopsis thaliana</i>: Total RNA Isolation and cDNA Synthesis</h2>
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- | <p align=justify><font>
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- | The frozen plant material has to be grinded in a precooled mortar in liquid nitrogen. About 120 mg of pulverized plant material are transfered into a precooled 2 ml Eppendorf tube and kept frozen until the following steps:<br>
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- | <ul>
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- | <li>Add 0.5 ml lysis buffer and immediately homogenize through rough shaking.</li>
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- | <li>Add 0.5 ml of saturated phenol and mix strongly.</li>
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- | <li>Add 0.5 ml of chloroform isoamyl alcohol (24:1) and vortex again at high speed for at least 30 seconds.</li>
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- | <li>Centrifugate for 5 min at 13,000 rpm.</li>
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- | <li>The lower phase contains now lipids and lipophilic compounds. The upper phase contains nucleic acids (~ 550 µl) and has to be carefully transferred into a new 2 ml Eppendorf tube. This tube has to be filled with 0.5 ml saturated phenol and 0.5 ml chloroform isoamyl alcohol (24:1). Mix immediately.</li>
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- | <li>Centrifugate at 13,000 rpm for 3 minutes.</li>
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- | <li>Prepare a new 2 ml Eppendorf tube with 1 ml of chloroform isoamyl alcohol (24:1). Transfer the upper aqueous phase (~ 540 µl) containing the protein purified nucelic acids into the new tube and vortex strongly.</li>
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- | <li>Centrifugate at 13,000 rpm for 3 minutes.</li>
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- | <li>Prepare a new 1.5 ml Eppendorf tube with 0.5 ml of pure isopropanol. For the last time transfer the upper phase (~ 400 µl) into the new tube and mix gently.</li>
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- | <li>Incubate the mixture over night at -20°C. The nucleic acids will precipitate.</li>
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- | <li>Centrifugate the samples at 13,000 rpm for 15 minutes at 4°C.</li>
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- | <li>Discard the supernatant and resuspend the pellet in 375 µl sterile H<sub>2</sub>O.</li>
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- | <li>Add 125 µl 8 M lithium chloride and incubate for 2 hours on ice at 4°C. At this point most of the RNA is going to be precipitated.</li>
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- | <li>Centrifugate at 13,000 rpm at 4°C and discard the supernatant.</li>
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- | <li>Wash the pellet with 100 µl 70% (v/v) ethanol and discard it after centrifugation.</li>
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- | <li>Dry the pellet at room temperature.</li>
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- | <li>Dissolve the pellet in sterile H<sub>2</sub>O (~ 25 µl, depending on the size of the pellet).</li>
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- | <li>Check the quantity and quality of the RNA with a Nanodrop spectrophotometer before starting with a cDNA synthesis.</li>
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- | </ul>
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- | </font></p>
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- | </html>
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In this section we are going to note all protocols that the teams have used for their work.