CHAPTER 3 METHODS
3.2 DNA isolation
3.1.6.2 Preparation of inoculum and inoculation of test plate
Performed according to section 3.1.4.2
3.1.6.3 Culture, incubation and reading of results
Performed according to section 3.1.4.3
harvested from the surface of Middlebrook 7H11 agar media with a sterile plastic loop and suspended in a sterile, round-bottomed 2 ml Eppendorf tube prefilled with 500 µl of sterilize distilled water. The tubes were then closed and placed for 30 minutes in a heating-block set at 80 oC to heat-kill the live bacteria. Next the tubes were transferred to an Eppendorf thermomixer set at 60 oC and 70 µl 10%
SDS and 50 µl proteinase K (10 µg/ml) were added to it. The Eppendorf thermomixer was set at low shaking mode i.e. 10 second periods of mixing alternating with 10 second periods of no mixing. The Proteinase K is used to both digest protein to release DNA from cells and to inactivate DNases that may degrade DNA. The action of Proteinase K is potentiated by SDS. After one hour 100 µl 5M NaCl and then 100 µl 10% CTAB, both preheated to 60 oC, were added to each tube and thoroughly mixed by inverting the tube by hand. The tubes were then subjected to another 15 minutes in the Eppendorf thermomixer set at 60 oC together with low-shaking mode. CTAB is a surfactant that, together with NaCl binds to and remove polysaccharides from lysed bacterial suspensions192. To purify the DNA further, 700 µl of a chloroform:isoamyl alcohol (24:1) mixture was added to all the tubes and mixed by inverting 20 to 25 times by hand. This led to the formation of a homogenous white “milky” solution. The tubes were then centrifuged for 10 minutes at 15625.503 x g and the upper aqueous phase (±700 µl) transferred to sterile microcentrifuge tubes containing 700 µl cold isopropanol (Sigma-Aldrich, SA). It was then carefully mixed by inverting the tubes several times so that the precipitated DNA could be seen as a thin thread. All the tubes were then kept overnight at 4 oC and then centrifuged at 9245.86 x g for 10 minutes at 4 oC. Thereafter the isopropanol was decanted and each pellet washed with 50 µl 80% cold ethanol (Merck, SA) followed by centrifugation for a
further 5 to 10 minutes at 13314.038 x g. This washing step was repeated once and the pellet left to dry by evaporation for 30 minutes at room temperature, by placing each opened tube upside-down on a clean paper towel. The heat-killed, isolated and cleaned DNA was then kept at 4 °C in 55 µl of 1x TE buffer until use.
3.2.3 Estimation of DNA purity, concentration and quality
DNA purity was estimated with the A260/A230 and A260/A280 absorbance ratios using the NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, Massachusetts, USA) and DNA quality was visualized by electrophoresing 1 µl extracted DNA in a 1% agarose gel (Appendix A.20)174,194.
Nucleotides, DNA and RNA all absorb at a wavelength of 260 nm and most other expected substances in molecular samples at either higher or lower wavelengths.
By also measuring absorbance at 230 nm and 280 nm, contaminants can be excluded by looking at A260/A280 and A260/A230 absorbance ratios. The A260/A280 and A260/A230 absorbance ratios were therefore used to evaluate the purity of DNA.
With the A260/A280 ratio, “pure” DNA is expected to have a A260/A280 absorbance ratio of ±1.8 and a A260/A230 absorbance ratio of ±2.0-2.2. If the ratios are considerably lower than these values, then it may indicate the presence of substantial amounts of impurities that absorb light at either 280 nm or 230 nm.
Two microliters of each DNA sample were therefore loaded onto the absorbance platform in order for the instrument to measure absorbance194.
Gel electrophoresis was used to assess DNA quality. First, a 1% agarose gel was prepared. A casting tray and 20-well plastic comb were cleaned with 70% alcohol followed by the proper positioning of the plastic comb inside the casting tray. The open ends of the tray were then secured with masking tape to prevent the gel from leaking out of the tray. Next 1X Tris-borate-EDTA (TBE) buffer was freshly prepared by adding 100 ml of 10X TBE to 900 ml distilled water (Appendix A.21).
The final 1% agarose solution was prepared by adding 1.4 grams of agarose (Seakem LE Agarose, Whitehead Scientific, SA) to 140 ml of the 1X TBE buffer.
The agarose was then dissolved in the 1X TBE buffer by heating it in a microwave oven and the full amount was then poured into the casting tray once it cooled down to approximately 40 to 45 oC. It was then left for 30 to 40 minutes to solidify at room temperature (25 oC) after which the comb and masking tape were
removed. The gel was then carefully placed in an electrophoresis tank and 1 X TBE buffer was added to the tank so that its surface was covered with
approximately 1-2 mm of fluid.
For each DNA sample, 5 µl of gel loading dye (Appendix A.22) was first dispensed onto a sheet of parafilm. Next the DNA was mixed inside its tube by tapping it lightly with a finger a couple of times and then 1 µl of the DNA was transferred directly into the drop of gel loading dye. The two were then mixed by pipetting up and down 3 to 4 times. The resultant 6 µl drop was immediately transferred to its appointed well in the gel, before continuing with the next sample. The last well was loaded with 5 µl gel loading dye together with 1 µl of a DNA molecular weight marker (DNA Molecular Weight Marker II, Roche, SA). The ladder served as a
molecular weight marker, so that the size of DNA fragments in the gel could be determined.
The gel was then covered with a lid so that the negative (black) cathode was positioned closest to the DNA samples in their wells and the positive (red) anode positioned furthest away from the DNA samples. DNA samples then migrated toward the positively charged electrode. The electrical power source was set at 100 Volts and allowed to run for one hour. The current remained between 35 and 50% of the Volts for the duration of the experiment. Next the gel was removed from its tank and placed inside the Syngene G:Box gel imaging system for image capturing and printing with the GeneSnap software package (Syngene, Maryland, USA). The size and brightness of bands were compared with that of the molecular weight marker as a rough estimate of the quality and quantity of the DNA.
3.3 Genotyping using IS 6110 restriction fragment length polymorphism