1.Lysis buffer: 0.5 M NaCl, 1% sodium dodecyl sulfate (SDS), 10 mM Tris–HCl, pH 8, 1 mM EDTA, pH 8. Store at room temperature. Heat at 65°C before using to dissolve the SDS.

2.Phenol:chloroform: isoamylalcohol. 25:24:1 (PCI): 25 ml phenol equilibrated with Tris–HCl, pH 8; 24 ml chloroform (equilibrated with Tris–HCl, pH 8), 1 ml isoamylalcohol. Store under 0.1 M Tris–HCl, pH 8, in a tightly capped, dark glass bottle at 4°C. Before using, check that the PCI is colourless. Discard and make up a fresh batch if any hint of colour is detected.

3.Chloroform:isoamylalcohol 24:1 (CI): 24 ml chloroform (equilibrated with Tris–HCl, pH 8), 1 ml isoamylalcohol. Store in a tightly capped, dark glass bottle at 4°C.

4.T0.1E buffer: 10 mM Tris–HCl, pH 8, 0.1 mM EDTA, pH 8. Store at room temperature.

1. Sample preparation is the most crucial step in ITC. The initial quantities and masses of ITS rDNA motif and ZAP1 DBD were noted

2. 0.1M Sodium Phosphate buffer(pH 7.4) is prepared from a stock solution of 55mM Sodium Phosphate and used as standard ITC buffer after taking the advice of P.hD in structural biology, Kunal Dhankar[1]. This was done as both DNA and protein are relatively large and act as macromolecules and thus remain stable in the phosphate buffer.

3. A stock solution of Protein DBD, which was ordered from GenScript, was produced. Protein with a concentration of 50 μM was prepared in the 0.1M phosphate buffer

4. Stock solution of ITS rDNA Motif Site, which was ordered from IDT DNA, was produced. DNA with a concentration of 500 μM was prepared in the same buffer initially

1. The Malvern Microcal PEAQ-ITC machine was washed first using deionised water 3 times to ensure there was no contamination in the experiment.

2. This was followed by washing using the phosphate buffer to allow proper loading of macromolecules.

3. All stock solutions were maintained at a pH of 7.4 to maintain stability.

1. 280μL of protein stock solution (50 μM) was loaded in the primary cell of ITC. 40 μL of the ITS rDNA Stock solution(500 μM) was loaded in a specialized ITC syringe and loaded into the sample cell of ITC.

2. 280 μL MilliQ water was kept in the reference cell during the titration.

3. Reference power was set at 10 kcal/min, and stir speed was kept at 750 rpm.

4. A total of 19 ligand injections were done in the sample cell with 1st injection of 0.4ul followed by 18 injections of 2ul.

5. Titration was recorded and fitted into the software provided by Malvern.

6. Ligand concentration was adjusted according to the results obtained to get the best possible fit. Values of n, ΔG, ΔH, and TΔS were determined.

Data analysis would be done using the MicroCal PEAQ-ITC analysis software.

1. Day culture: ~10 mL fresh liquid F’ E. coli DH5aplha culture, roughly log-phase

2. Molten (~48-52 ˚C) agarose top, 3 mL per sample

3.100 (or 50) µl aliquot competent cells

4. 10-15 mL sterile culture tubes, e.g. VWR #47729-568, 17 mm x 100 mm tubes

5. SOC recovery medium, at room temperature, or ideally, 37 ˚C

6. LB/IPTG/XGal plates, pre-warmed 1 hr 37 ˚C

7. M13 RF DNA, freshly diluted in water to 1-5 ng/µl (NEB #E8101S contains M13KE at 1 µg/µl)

8. 2 mm (or 1mm) electroporation cuvette, on ice, e.g. BTX #45-0134, #54-0135

1. Remove electrocompetent cells from the freezer and place them on ice. Immediately, add 1-5 ng M13 RF DNA to the surface of the frozen cell pellet. Thaw on ice for approximately 5-10 minutes. Finger flick tube to confirm thaw and mix cells and DNA. Transfer cells to a chilled cuvette without introducing bubbles. Tap the cuvette on a hard surface to bring cells to the bottom and eliminate air bubbles.

2. Electroporate using the following conditions for Bio-Rad GenePulser electroporators: 2.5 kV, 200 Ω, and 25 μF (for 1 mm cuvette/50 µl aliquot:1-5 ng DNA, 2.1 kV, 100 Ω, and 25 μF)

3. Immediately add 1 mL of 37°C SOC to the cuvette, then transfer to the culture tube. Shake vigorously (250 rpm) or rotate at 37 °C for 30-45 min.

4. Prepare 103 and 104 dilutions of the outgrowth. Transfer 10 μl outgrowth dilution to a sterile tube of 200 μl day culture cells and 3 mL agarose top. Briefly mix by vortexing for a few seconds. Pour onto the pre-warmed LB/IPTG/XGal plate. Gently tilt and rotate the plate to spread to agarose evenly. Incubate plates overnight at 37°C.

LB Medium:
10 g Bacto-Tryptone, 5 g yeast extract, 5 g NaCl. Autoclave, store at room temperature.

Per litre: 10 g Bacto-Tryptone, 5 g yeast extract, 5 g NaCl, 7 g electrophoresis grade agarose. Dispense into 100-200 mL aliquots. Autoclave and microwave, as needed.

Mix 1.25 g IPTG (isopropyl-β-D-thiogalactoside) and 1 g Xgal (5-Bromo-4-chloro-3-indolyl-β-D-galactoside) in 25 ml DMF (dimethyl formamide). The solution can be stored at –20°C.

Per liter: liter LB medium, 15 g agar. Autoclave, cool to < 70°C, add 1 ml IPTG/Xgal Stock per liter and pour. Store plates at 4°C in the dark.

Per liter: 20 g Bacto-Tryptone, 5 g yeast extract, 0.5 g NaCl. Dispense into 100 ml aliquots, autoclave and then store at room temperature. Prior to use, add 0.5 ml 2 M MgCl2 and 2 ml 1 M glucose (both sterile) per 100 ml. Then store at 4°C.

1.Fungal Genomics: Methods and Protocols: Manfred G. Grabherr, Evan Mauceli, Li-Jun Ma (auth.), Jin-Rong Xu, Burton H. Bluhm (eds.)

2.Biolabs, N.E. Typical lamp protocol (M0275),

3.Yasuyoshi Mori 1 et al. (2002) Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation, Biochemical and Biophysical Research Communications.

4.Biolabs, N.E. Transfection of M13 DNA protocol, NEB. Available at: