2.2 Methods
2.2.8 Isolation of nucleic acids
(i) Small-scale isolation of plasmid DNA from bacterial culture
Small-scale or minipreparation of plasmid was made by alkaline lysis with SDS (Sambrook and Russell 2001). Briefly, 5 ml of overnight bacterial culture was centrifuged in microfuge tube for 2 min at 6000 rpm. Pellet was resuspended in 100 µl alkaline lysis
solution II was added to the bacterial suspension, contents were mixed by inverting the tubes 4-5 times and kept on ice for 10 min. To the viscous bacterial lysate, 150 µl alkaline lysis solution III was added, mixed by inverting the tube and kept on ice for 3-5 min. The tube was centrifuged for 10 min at 12,000 rpm and 4°C. The supernatant was transferred to the fresh microfuge tube and equal volume of Phenol:chloroform:isoamyl alcohol (25:24:1) was added, centrifuged for 10 min at 12,000 rpm and 4°C. The aqueous phase was taken in a fresh microfuge tube (1.5 ml) and plasmid DNA precipitated by adding 1.5 volumes of absolute ethanol. The tube was gently inverted few times and the DNA was pelleted by centrifuging the tube for 10 min at 12,000 rpm. The supernatant was discarded and the pellet was washed with 70% ethanol by centrifuging for 2 min at 12,000 rpm. The traces of ethanol was completely removed and the DNA pellet was allowed to dry at room temperature for 15 min. Finally, the pellet was dissolved in about 30 l of 1X TE buffer (pH 8.0) and stored at 4ºC.
(ii) Large scale isolation of plasmid DNA from bacterial culture
Large-scale preparation of plasmid DNA (~500 µg) was made by alkaline lysis method (Sambrook and Russell 2001). Briefly, centrifuged overnight (250 ml with antibiotic) at 6,000 rpm for 10 min. The pellet was suspended in 9 ml of alkaline lysis solution I, 1 ml of a freshly prepared solution of 10 mg/ml lysozyme and 20 ml of freshly prepared alkaline lysis solution II. The contents were thoroughly mixed by gently inverting the bottle several times and incubated for 5-10 min at room temperature. 10 ml of alkaline lysis solution III was added to the contents and gently mixed by swirling several times and placed the mixtures on ice for 5-10 min. The bacterial lysate was centrifuged for 15 min at 12,000 rpm at 4°C. The supernatant was transferred to the fresh tube and 0.6 volume of isopropanol was added and stored the tube at room temperature for 10 min. The precipitated nucleic acid was recovered by centrifugation at 12,000 rpm at room temperature and washed once with 70% ethanol.
The pellet was dried at room temperature and then dissolved in 200 µl of TE buffer. RNAase A treatment was given to the plasmid solution and then stored at -20°C for further use.
(iii) Neurospora genomic DNA isolation
The strain of interest was grown in liquid Vogel’s glucose medium at 30°C for 2 to 3 days with shaking at 200 rpm. The mycelial mass was harvested by vacuum filtration and lyophilized. The dried mycelia were ground with glass beads (0.2 µm in diameter) using a
mortar and a pestle to a fine powder. Approximately, 150 mg of the powdered mycelia was taken in a 1.5 ml microfuge tube and 1 ml of lysis buffer added to it. Complete mixing of the mycelia and lysis buffer was achieved using a pipette tip or a toothpick. The tube was incubated at 65°C for 30 min, followed by centrifugation at 15,000 rpm for 10 min. The supernatant was taken in a fresh microfuge tube and 500 l of phenol:chloroform:isoamyl alcohol mixture (25:24:1) was added. The tube was rotated in a cell mixer for 15 min and centrifuged at 15,000 rpm for 10 min. The aqueous phase was carefully removed and the phenol:chloroform:isoamyl alcohol treatment repeated. The aqueous phase was taken in a fresh microfuge tube and washed with 600 l of chloroform to remove the last traces of phenol. The aqueous phase was taken in a fresh tube and genomic DNA precipitated by adding 1.5 volumes of absolute ethanol. The tube was gently inverted few times and the genomic DNA pelleted by centrifuging the tube for 10 min at 15,000 rpm. The supernatant was discarded and the pellet was washed with 70% ethanol by centrifuging for 2 min at 15,000 rpm. The traces of ethanol were completely removed and the genomic DNA pellet was allowed to dry at room temperature for 15 min. Finally, the pellet was dissolved in about 60 l of 1X TE buffer (pH 8.0) and stored at 4ºC. All centrifugations were carried out at 25ºC.
(iv) Neurospora RNA isolation
The conidia of the strain of interest were grown in 250 ml conical flasks in liquid Vogel’s glucose medium at 30°C and 150 rpm for 12 to 16 h. The mycelial mass was harvested by vacuum filtration and immediately frozen in liquid nitrogen. The frozen tissue was ground to a fine powder using a mortar and a pestle. The powder was immediately transferred into a 2 ml microfuge tube containing 0.3 ml TRIzol reagent (Invitrogen, CA) to protect RNA from degradation by RNAase followed by further addition of the mixture of 0.75 ml lysis buffer (0.6 M NaCl, 10 mM EDTA, 100 mM Tris HCl, pH 8.0, 4% SDS) and 0.75 ml phenol (saturated with 0.1 M Tris HCl, pH 8.0). The tube was rotated in a cell mixer for 20 min and centrifuged at 10,000 rpm for 10 min. The upper aqueous phase was carefully removed and transferred to a fresh 2 ml microfuge tube containing an equal volume of phenol (saturated with 0.1 M Tris HCl, pH 8.0). The mixture was vortexed for few seconds and centrifuged for 10 min at10,000 rpm. The upper aqueous phase was transferred to a fresh 2 ml microfuge tube and 0.75 ml of 8 M LiCl was added. The mixture was stored overnight at 4°C for 16-20 h. The next day mixture was vortexed briefly and centrifuge for 10 min at
10,000 rpm. The pellet was resuspended, which is not always visible, in 0.3 ml double distilled water, mixed with 0.03 ml 3 M Na-acetate (pH 5.2) and 0.75 ml ethanol. The mixture was stored at -20°C for 2 h and centrifuged for 10 min at 10,000 rpm. The supernatant was discarded and the precipitate was washed with 70% ethanol. The RNA pellet was dried in room temperature for 10 to 15 min and re-dissolved in DEPC treated water. The RNA solution was stored at -70°C. The next day RNA sample were analysed in 1.2% agarose gel containing 2.2 M formaldehyde and 1X MOPS solution. Purity of the RNA preparations was assayed by spectrophotometric measurements. The A260/A230 and A260/A280 ratios were 2 or more, indicating the absence of any protein or polysaccharide contamination.