Un criterio de buen gobierno

Genomic DNA of C. albicans clinical isolates was digested with restriction enzymes and separated by gel electrophoresis, stained and visualized as described in section 2.8.10. DNA fragments were transferred to positively charged nylon membranes using a modification of the method of Southern (Ausubel et al., 2006). Briefly, the gels were prepared for blotting as follows: The initial step was depurination in 0.25 M HCl for 15 min, followed by denaturation in 0.5 M NaOH plus 0.5 M NaCl for 2 x 20 min and lastly neutralization in 2M NaCl, 0.5 M TrisCl, pH 7.4 for 2 x 30 min, under gentle agitation. After neutralization, the gel was washed for 2 min in 2x SSC and assembled onto a blotting apparatus which as follows: a glass plate was placed across a glass tray containing 200 ml of 20x SSC buffer. A 3 MM paper wick soaked in 20x SSC was placed on the top of the glass, covered with plastic wrap, which was cut to have a surface area slightly smaller than the gel. The gel was placed on top of the soaked paper wick with the DNA side facing the top. A nylon membrane cut to the same size as the gel was pre-soaked in 2x SSC and then placed on top of the gel; three sheets of 3 MM paper of the same size as the gel were pre-soaked in 2x SSC and placed on top of the membrane, followed by a stack of paper towels and a 500 g weight. The blot assembly was left overnight to allow DNA to transfer from the gel to the membrane. After transfer, the membrane was washed in 2x SSC for 5 min and DNA was crosslinked to the membrane using an Ultraviolet crosslinker Cex-800 (Ultra-Lum Inc) for 5 min (120,000 IJ/cm3).

2.8.12.1. Probe labeling using Dig-labeling system

The DNA probes used in Southern blot analysis were labeled using PCR DIG Probe Synthesis Kit (Roche), and the efficiency of probe labeling was evaluated, as per the manufacturer’s instructions.

2.8.12.2. Hybridization

The membranes were prehybridized in hybridization buffer, DIG Easy Hyb (Roche), for 2 hours at 42oC in glass hybridization tubes (Amersham Biosciences). The labeled probes were denatured by boiling for 10 min, followed by cooling on ice for 5 min, and added to the hybridization tubes. The final concentrations of the probes were adjusted to 12-15 ng/ml according to the efficiencies of labeling determined as described in section 2.8.12.1. Hybridizations were carried out at 42°C for 16 h according to the manufacturer’s instructions (Roche). Afterwards the blots were washed and probe-target hybrids detected with the Anti-Digoxigenin-AP kit (Roche) in detection buffer containing CSPD (Roche), according to the manufacturer’s instructions (Roche). The blots were then exposed to X-ray film (Kodak) at 37oC for 30 min. Films were developed in a 100PlusTM automatic X-ray processor (All- Pro Imaging Corp.).

2.8.13. DNA sequencing

DNA sequencing was performed by the Allan Wilson Centre Genome Service

(AWCGS), Massey University. Samples for sequencing were prepared as per the

facility’s instructions: the reaction mixture of 15 µl contained 300 ng of plasmid

DNA or 2 ng/100 bp of PCR products and 3.2 pmol of each sequencing primer. The

samples were sent to AWCGS and were sequenced by the dideoxynucleotide chain termination method using BigDyeTM Terminator Version 3.1 Ready Reaction Cycle Sequencing Kit on the capillary ABI3730 Genetic Analyzer, from Applied Biosystems Inc. Sequences. Electrophoregrams were assembled using AB DNA Sequencing Analysis Software version 5.2. (Applied Biosystems Inc.).

2.9. PCR methods

Oligonucleotide primers used in this study are listed in Table 2.7. All primers were synthesized by Invitrogen. Primers were re-suspended in sterile deionized water to a concentration of 100 pmol/µl (stock solution) and stored at -20oC. Primers used in PCR reactions were diluted to a concentration of 10 pmol/µl (working solution).

2.9.1. Routine PCR

Routine polymerase chain reactions (PCRs) were performed in a final volume of 20

µl containing 1 U of Qiagen Taq DNA polymerase, 4 µl of Q-buffer and 1x PCR buffer supplied by the manufacturer (Qiagen), 10 pmol of each primer, 200 µM of

primer sets and the size of the products (Ausubel et al., 2006) and included an initial incubation for 2 min at 94°C, followed by 30 cycles of 45 s at 94°C, 45 s at 50– 60°C, and 30 s to 3 min at 72°C. All PCR protocols included a final 5 min extension step at 72°C. Reactions were carried out in an Eppendorf Mastercycler thermocycler (Eppendorf, Hamburg, Germany).

2.9.2. Colony PCR

Colony PCR was performed as described in Zhang et al. (2010). Briefly, a portion of a C. albicans colony or E. coli colony was picked with a sterile 10 µl pipette tip and mixed with 20 µl PCR reaction mixture prepared as described above; the initial step in the cycling program was altered to 5 min (for C. albicans) or 3 min (for E. coli) at 96°C.

2.9.3. High-fidelity PCR

High-fidelity enzyme KOD DNA Polymerase (Novagen) was used, according to the manufacturer’s instructions, for amplifying fragments used in constructing resistance markers where sequence accuracy was crucial.

2.9.4. Recombinant PCR

Recombinant PCR was performed as described by Zarrin et al. (Zarrin et al., 2005). In short, two rounds of PCR reactions were performed (Fig. 2.2). For first-round PCR, fragment 1 and 2 were amplified separately with primers p1 and p2, and p4

and p5, respectively. Each reaction was performed in a final volume of 50 µl containing 1 U of KOD DNA polymerase (Novagen), 5 µl of 10x PCR buffer supplied by the manufacturer (Novagen), with 10 mM MgCl2, 20 pmol of each primer, 200 µM of each dNTP, and 10–100 ng DNA. Hot start amplification was initiated with 2 min at 94 °C denaturation, followed by 30 amplification cycles (94 °C for 30 s, 50 °C for 30 s, and 72 °C for 1 min) and a final extension cycle of 72°C for 5 min. PCR products from each reaction were gel-purified using a Zymoclean™ Gel DNA Recovery Kit(Zymo Research) and used in a subsequent amplification. Second-round PCR was as for the first-round, except that 10 ng each of fragment 1 and fragment 2 PCR product from the first round of PCR, together with 20 pmol of primers p1 and p5, 2 pmol of primer p3 were used. PCR conditions were as for the first-round of PCR, except that the extension time at 72 °C was 2.5 min. PCR product was gel-purified using a Zymoclean™ Gel DNA Recovery Kit (Zymo Research).