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{{Team:Bielefeld/Head}} <center> =This page lists all molecular genetics protocols we use in our project= </center> ==Yeat: Complete genome isolation== The complete genome isolation was done with the Promega Wizard genomic DNA purification system kit. <div style="text-align:justify;"> *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. *Remove the supernatant. *Resuspend the cells in 90 μL of 50 mM EDTA. *Add 10 μL of 1000u lyticase and pipet 4 times to mix. *Incubate the sample at 37°C for 60 minutes to digest the cell wall. *Centrifuge the sample at 14,000 × g for 2 minutes and then remove the supernatant. *Add 300 μl of Nuclei Lysis Solution to the cell pellet and pipet to mix. *Add 100 μl of Protein Precipitation Solution and vortex at high speed for 20 seconds. *Let the sample sit on ice for 5 minutes. *Centrifuge at 14,000 × g for 3 minutes. *Transfer the supernatant containing the DNA to a clean 1.5 ml tube containing 300 μl of room temperature isopropanol. *Gently mix by inversion until the DNA is visible. *Centrifuge at 14,000 × g for 2 minutes. *Carefully decant the supernatant and drain the tube on clean absorbent paper. *Add 300 μl of room temperature 70% ethanol and invert the tube several times to wash the DNA pellet. *Centrifuge at 14,000 × g for 2 minutes. *Drain the tube on clean absorbent paper and allow the pellet to air-dry for 15 minutes. *Add 50 μl of DNA Rehydration Solution. *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. *Rehydrate the DNA by incubating at 65°C for 1 hour. Periodically mix the solution by gently tapping the tube. *Store the DNA at 2–8°C. ==''Arabidopsis thaliana'': Growth Conditions and Plant Material== Six weeks old ''A. thaliana'' 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 &#91;100 &micro;mol &frasl; quanta m^-2s^-1&#93; 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 &#91;100 &micro;mol &frasl; quanta m^-2s^-1&#93; 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. ==''Arabidopsis thaliana'': Total RNA Isolation== 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: *Add 0.5 ml lysis buffer and immediately homogenize through rough shaking. *Add 0.5 ml of saturated phenol and mix strongly. *Add 0.5 ml of chloroform isoamyl alcohol (24:1) and vortex again at high speed for at least 30 seconds. *Centrifugate for 5 min at 13,000 rpm. *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. *Centrifugate at 13,000 rpm for 3 minutes. *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. *Centrifugate at 13,000 rpm for 3 minutes. *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. *Incubate the mixture over night at -20°C. The nucleic acids will precipitate. *Centrifugate the samples at 13,000 rpm for 15 minutes at 4°C. *Discard the supernatant and resuspend the pellet in 375 µl sterile H₂O. *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. *Centrifugate at 13,000 rpm at 4°C and discard the supernatant. *Wash the pellet with 100 µl 70% (v/v) ethanol and discard it after centrifugation. *Dry the pellet at room temperature. *Dissolve the pellet in sterile H₂O (~ 25 µl, depending on the size of the pellet). *Check the quantity and quality of the RNA with a Nanodrop spectrophotometer before starting with a cDNA synthesis. ==''Arabidopsis thaliana'': cDNA Synthesis== After a successful total RNA isolation the RNA has to be translated in cDNA through RT-PCR: *Take 3 µg/µl of total RNA and add sterile H₂ to 8 µl. Additionally add <table border="0" rules="cols" align="center"> <tr> <td align=center> 1,1 mM </td> <td align=center> Oligo-d(T)-Primer </td> </tr> <tr> <td align=center> 0,83 mM </td> <td align=center> dNTPs </td> </tr> <tr> <td align=center> 3,5 µl </td> <td align=center> H₂O </td> </tr> </table> *Vortex and centrifugate shortly. *Incubate the samples for 10 minutes at 70°C. *Immediately transfer the samples into ice water for 5 minutes. *After cooling the samples centrifugate shortly. *To start the synthesis add <table border="0" rules="cols" align="center"> <tr> <td align=center> 6 µl </td> <td align=center> 5xMMLV-Puffer </td> </tr> <tr> <td align=center> 4,5 µl </td> <td align=center> H₂O </td> </tr> <tr> <td align=center> 1 µl </td> <td align=center> MMLV-reverse Transkriptase [200 U/µl] </td> </tr> <tr> <td align=center> 0,5 µl </td> <td align=center> RNasin RNase-Inhibitor [40 U/µl] </td> </tr> </table> *Mix the samples and centrifugate shortly. *Incubate for 1 hour at 42°C to translate the RNA into cDNA. *Transfer the samples to 70°C for 15 minutes to stop the reaction. *The new synthesized cDNA can be used for PCR after diluting 1:10 with water. Store the cDNA at -20°C. ==Ethanol precipitation to clean DNA== To get rid of distracting salts the DNA has to be cleaned. For this we used the following protocol: *If the volume of the sample containing the DNA is less than 200 µl bring the volume up to 200 µl. *Add 1/10th volume of 3M sodium acetate and mix. *Now add 2 volumes of -20°C cold 100% ethanol and vortex for 10 seconds. *The sample can now be placed in a -20°C freezer overnight or incubated for 30 minutes at -80°C. *Centrifugate for 10 minutes at 4°C. *Discard the supernatant containing the ethanol. *Wash the pellet with 500 µl 4°C cold 70% ethanol by rolling the sample gently. *Discard the supernatant. *Let the pellet dry at room temperature or speedvac the pellet. *Resuspend the Pellet in water (amount is depending on the size of the pellet).

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