DNA extraction, quantification and analysis

2.2.1 Genomic DNA isolation from P. arachidicola by CTAB method

The CTAB (hexadecyltrimethylammonium bromide) DNA extraction method used in this research was developed by Doyle and Doyle (Doyle and Doyle, 1987). This method allows a high yield of high molecular weight genomic DNA from fresh freeze dried fungal samples. P. arachidicola is a very slow growing fungus and further experimentation requires high molecular weight genomic DNA, therefore this method was chosen.

The freeze-dried mycelia were ground using a sterile mortar and pestle with liquid nitrogen. 500 mg of ground sample was transferred into a sterile microcentrifuge tube. 600 μl CTAB was added to the sample and mixed thoroughly by inversion. The sample was incubated at 37°C for 10 minutes and then at 65°C for 45 minutes with occasional inversion. Removed from the water bath, the sample was cooled to room temperature and 600 μl chloroform was added, mixed by inversion then stored on ice for 2 minutes. After centrifugation at 15110 g (13000 rpm Biofuge) for 2 minutes, the upper aqueous phase was transferred to a new tube and 600 μl isopropanol was added, mixed and stored on ice for 5 minutes. The mixture was centrifuged at 15110 g (13000 rpm Biofuge) for 30 seconds to settle the DNA, the solution was discarded, 600 μl 80% ethanol was added to the precipitated DNA and centrifuged at 15110 g (13000 rpm Biofuge) for 1 minute. The ethanol washing step was repeated at least twice. Then the ethanol was decanted off completely, and the sample was left to air dry. Finally the DNA was resuspended in 50 μl TE buffer.

2.2.2 Purification of P. arachidicola genomic DNA

Two purification methods, phenol purification and column purification were used. The column purification method, involves using a commercial kit which was convenient, did not involve hazardous chemicals and can yield high quality purified genomic DNA. But the purified genomic DNA was slightly fragmented (around 50 kb), which is not suitable for 8-cutter Southern blot. So the phenol purification method, which can yield high molecular weight genomic DNA was used when larger fragments of gDNA were required.

2.2.2.1 Column purification

The extracted genomic DNA was purified using QIAquick Gel extraction kit (Qiagen) according to the manufacturer’s instruction.

2.2.2.2 Phenol purification

To unpurified genomic DNA sample, an equal volume of phenol and equal volume of chloroform was added, mixed thoroughly by vortex then centrifuged at 15110 g (13000 rpm Biofuge) for 3 minutes. The upper aqueous phase was transferred to a fresh microcentrifuge tube and an equal volume of chloroform was added, mixed thoroughly by vortex and centrifuged at 15110 g (13000 rpm Biofuge) for 1 minute. The DNA was then precipitated by transferring the upper aqueous phase to a new microcentrifuge tube and adding 0.1 volume of 3 M Na acetate and 0.6 volume of isopropanol to the sample. The tube was inverted several times to mix the sample and centrifuged at 15110 g (13000 rpm Biofuge) for 5 minutes to settle the DNA. The supernatant was discarded and an equal volume of 70% ethanol was added to the sample, then centrifuged at 15110 g (13000 rpm Biofuge) for 5 minutes. The ethanol was completely decanted, the sample left to air dry and the DNA resuspended in water. The purified DNA was stored at 4 °C.

2.2.3 Isolation of plasmid DNA

Plasmid DNA was isolated using an Ultra-fast rapid alkaline extraction method 16 

(Cormack and Somssich, 1997). 0.2 ml of overnight E. coli culture (section 2.1.1) was transferred into a sterile microcentrifuge tube. 0.2 ml of lysis solution (1% SDS, 0.2 N NaOH) was added to the culture and mixed by inverting the tubes several times. Immediately, 0.2 ml of solution III (3 M potassium acetate, pH 5.50) was added to the tube and mixed gently by inverting the tube. After centrifugation at 15110 g (13000 rpm Biofuge) for 1 minute, the supernatant was transferred into a fresh tube, 0.5 ml isopropanol was added and mixed by inversion. After centrifugation as before, the supernatant was discarded and the pellet left to air dry. 50 μl TE buffer was added to resuspend the pellet. This method can reduce the time of isolating plasmid DNA for further restriction analysis, but the quality of plasmid is not so good for sequence analysis. So to increase the yield and quality of plasmid DNA isolated from E. coli cells, QIAprep Spin Miniprep kit (Qiagen) was used according to manufacturer’s instructions.

2.2.4 Fluorometric assay to determine DNA concentration

DNA was quantified using a “Hoefer DyNA Quant 200 fluorometer” (Amersham Biosciences). 2 ml of working buffer (A2.7) was used to zero the fluorometer, then 2 μl of standard DNA (100 ng/ml) (A2.5) was added to 2 ml of working buffer in the cuvette for calibration. To determine the sample DNA concentration, 2 μl of sample DNA was added to 2 ml of working buffer, the concentration recorded by the fluorometer as ng/μl.

2.2.5 Agarose gel electrophoresis

An 0.4%-1% (w/v) agarose gel was prepared depending on the DNA sample molecular weight. 1x TBE buffer (A2.3) was added to the gel apparatus, the DNA sample was mixed with gel loading dye (A2.9) and loaded along with an appropriate DNA ladder on the gel. The electrophoresis was carried out at 40-80 volts until the dye moved to 2 cm from the end of the gel. Then the gel was stained with 1 mg/mL ethidium bromide staining buffer (A2.8) for 15 minutes followed by rinsing in MilliQ water for 2 minutes. Finally the gel was visualized and photographed using the Gel 17 

Documentation system (BioRad) and Quantity One 4.4.0 basic software.

2.2.6 Agarose gel purification of size fractionated DNA

After gel electrophoresis, the selected DNA fragment was cut out from the gel using a sterile scalpel under long wave UV light and transferred into a sterile microcentrifuge tube. The DNA was then recovered using a QIAquick gel extraction kit (Qiagen) according to manufacturer’s instructions. The concentration and size of recovered DNA sample was measured using Fluorometric assay (section 2.2.4) and gel electrophoresis (section 2.2.5)

2.2.7 Restriction endonuclease digestion of DNA

The selected restriction endonuclease and appropriate digestion buffer was added to a DNA sample in a microcentrifuge tube according to manufacturer’s instruction. The volume of the digestion mixture (20 μl to 50 μl) depended on the amount of DNA sample used, the temperature of incubation depended on the selected restriction endonuclease. The incubation time was varied from 1 hour to overnight. The digestion of the DNA sample was checked using gel electrophoresis (section 2.2.5).To increase the performance of the restriction enzyme (for 8-cutter Southern blot), bovine serum albumin (BSA) was added to the restriction digestion mixture in a final concentration of 0.1 mg/ml (Williams et al., 1996).

Table 2.1 Example of EcoRI digestion of gDNA for Southern blot and fractionated genomic library. In a microcentrifuge tube, the following were added:

gDNA (150 ng/μl) 13.3μl EcoRI (10 unit/μl) 2μl Buffer H (10x) 5μl MQ water 29.7μl

Mix well and incubated in 37 °C overnight