This page lists all molecular genetics protocols we use in our project
Complete genome isolation from yeast with the Promega Wizard genomic DNA purification system kit
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 [100 µmol ⁄ quanta m-2s-1] 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-2s-1] 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 and cDNA Synthesis
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.
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