DNA isolation methods

2.6.1 Plasmid DNA isolation

Reagents:

Solution I: (50 mM Glucose, 10 mM EDTA, 25 mM Tris pH 8.0) Solution II: (0.2 M NaOH, 1% (w/v) SDS)

80

Solution III: (3 M potassium acetate, 11.5% (v/v) glacial acetic acid)

Plasmid DNA was isolated from NB and GV3101 cells by a modification of the alkaline lysis method of Sambrook et al. (1989). Cells from ~1.5 mL (for NB) or 3 mL (for GV3101) overnight cultures were pelleted by centrifugation at 10000 x g for 30 s at room temperature. The supernatant was decanted and the cells re-suspended in 200 μL of solution I by running the base of the microfuge tube over the holes of a microfuge rack. The re- suspended cells were then lysed by gently inverting in 200 μL of solution II for ~10 s and neutralized with 200 μL of solution III. The protein, carbohydrates and genomic DNA were pelleted by centrifugation at 10000 x g for 10 min at 4°C and ~500 μL of the cleared lysate removed to a fresh microfuge tube where it was precipitated with 1 volume of isopropanol. The precipitated plasmid DNA was pelleted by centrifugation at 10000 x g for 10 min at 4 °C, washed with 700 μL of 70% (v/v) ethanol and air-dried for ~10 min. The DNA was then dissolved in 20 μL sterile water and stored at -20°C until needed.

2.6.2 Genomic DNA isolation

Fresh leaves from two different ecotypes (Ler-0 and Columbia) and their heterozygous plants were frozen in liquid nitrogen. Genomic DNA for HRM analysis and cloning was isolated by Slipstream automation (Plant & Food Research, Tennent Drive, Palmerston North, New Zealand) or by utilizing the previously described DNA extraction protocol of Dellaporta et al., (1983). The purity and quantity of isolated DNA was tested with Nanodrop ND-1000 Spectrophotometer. Pure DNA samples with A 260/280 values of ~1.8 and A 260/230 values of ~2.0-2.2 were selected for downstream analysis.

2.6.3 Nuclear enriched genomic DNA isolation

Reagents:

Extraction Buffer 1: (0.4M sucrose, 10 mM Tris-HCl pH 8.0, 10 mM MgCl2, 5 mM β-

mercaptoethanol [β –ME]) To make 100 mL: 20mL of 2M sucrose, 1 mL of 1M Tris-HCl pH 8.0, 1 mL of 1M MgCl2, 35 μL of 14.3M β –ME, made to volume with water and stored at 4

81

Extraction Buffer 2: (0.25 M sucrose, 10 mM Tris-HCl pH 8.0, 10 mM MgCl2, 1% Triton X-

100, 5 mM β –ME) To make 10 mL: 1.25 mL 2M Sucrose, 100 μl of 1M Tris-HCl pH 8.0, 100 μL of 1M MgCl2, 0.1 mL of 100% Triton X-100 and 3.5 μL of 14.3M β –ME made to

volume with water. β –ME was added before use.

Extraction Buffer 3: (1.7 M sucrose, 10 mM Tris-HCl pH 8.0, 2 mM MgCl2, 0.15% Triton X-

100, 5 mM β –ME ) To make 10 mL: 8.5 mL of 2M Sucrose, 100 μL of 1M Tris-HCl pH 8.0, 20 μL of 1M MgCl2, 15 μL of 100% Triton X-100 and 3.5 μL of 14.3M β –ME, made to

volume with water. β –ME was added before use.

TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0)

Tris-buffered phenol: 250 g phenol crystals dissolved in 100 mL water containing 0.25g (0.1% w/v to phenol) of hydroxy quinoline. 100 mL 0.5 M Tris-HCl (pH 8.0) was mixed for ~15 min with the phenol. After phase separation, the upper layer was discarded the procedure repeated with 0.1 M Tris-HCl and ~1 cm layer of the Tris left over the phenol and the phenol stored at 4°C in the dark. The pH of buffer saturated phenol should be ≤ 7.5 for DNA

extraction. PCI, Phenol:chloroform:isoamyl alcohol (25:24:1)

Nuclear enriched genomic DNA was isolated according to the procedure of Lutz et al. (2010). Leaf tissue (~200 mg FW) was harvested into 2 mL microfuge tubes then snap frozen in liquid nitrogen and stored at -80°C until needed. On the day of DNA isolation, the tissue was removed to a mortar filled with liquid nitrogen and crushed with a pestle. The tissue was then transferred to a 50 mL Falcon tube containing 25 mL of ice-cold Extraction Buffer 1 and vortexed. The solution (green coloured) was then filtered through two layers of Miracloth (Calbiochem-Novabiochem, La Jolla, CA, USA) into a fresh sterile 50 mL falcon tube on ice. If cellular debris was present in the filtrate the solution was filtered again. The filtrate was then centrifuged at 1940 x g for 20 min at 4ºC to pellet the organelles including chloroplasts, which made the pellet green in colour. The supernatant was then discarded and the organelle-containing pellet re-suspended in 1 mL of ice-chilled Extraction Buffer 2, which contained Triton X-100 for preferential elimination of chloroplasts. The re-suspended enriched intact nuclei were pelleted by centrifugation at 12 000 x g for 10 min at 4ºC. Then re-suspended in 300 μL of ice-chilled Extraction Buffer 3 (by gently pipetting up and down to remove all signs of clumps) and gently overlaid on 300 μL of chilled Extraction Buffer 3 present in a clean 1.5 mL microfuge tube. The nuclei were then pelleted by centrifugation at 14 000 x g for 1 h at 4 °C and re-suspended in 720 μl TE buffer.

82

Contaminating RNA was removed from the DNA by digesting with RNase (10

μL 5 mg ml-1 _{RNase, 16 μL 5M NaCl and 64 μL water) at 65ºC for 30 min. The RNase and}

other contaminating proteins were removed from the DNA by digesting with Proteinase K (12 μL 10 mg mL-1 _{Proteinase K, 80 μL 10% SDS) at 45ºC for 1 h. The DNA was further}

cleaned by adding 800 μL PCI to the DNA in a 2 mL microfuge tube, shaking the mixture for 2 min by hand and centrifuging for 2 min at 980 x g. The supernatant was transferred to a clean tube and the PCI step repeated. The supernatant was then transferred to a clean tube containing one volume of chloroform (to remove traces of phenol), the tube shaken for 2 min and centrifuged and the supernatant containing the DNA collected. The procedure was repeated and the DNA then removed and precipitated with ~1 volume of isopropanol and 1/10 volume 3M NaOAc. The DNA was pelleted by centrifugation at 980 x g for 30 min at 4ºC and the pellet washed twice with 70% ethanol. The DNA was air-dried at room temperature for 10 min and re-suspended in 50 μL TE. An aliquot (1 μL) of DNA was run on a 1% (w/v) agarose gel against lambda DNA of known concentrations to help quantify and look for degradation. DNA concentration was confirmed with a Nanodrop ND-1000 Spectrophotometer.