• 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

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

• 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

• 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).

PCR for A.thaliana laccase ampflification

Standard BioBrick Assembly

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