Yeast Growth in YPD Agar: To prepare YPD agar, mentioned in Table 1 nutrient ingredients in given concentration were weighed and added to 200 ml of distilled water. The mixture was autoclaved (SMS ASL80 MSV) for 1.5 hours at 121°C. On sterile plates, 25-30 ml of the mixture was poured and left to cool down. The yeast cells were then streaked on the agar plates and the cultures were grown in a stationary incubator (S1-600R, Lab Companion) for 72 hours at 30°C. Yeast Growth in YPD: To prepare YPD liquid medium, glucose, peptone and yeast extract were weighed and added to 200 ml of distilled water. The mixture was autoclaved for 1.5 hours at 121°C. Then a loop of yeast biomass from single cells grown on YPD agar was used to inoculate 5 ml of YPD. …show more content…
S. Hoffman and F. Winston. 2 ml of the ON cultures were placed in Eppendorf tubes and spun for 2 min at maximum speed. The supernatant was discarded and 2 ml of each culture was added to the Eppendorf tubes, then spun again for 2 min at maximum speed and then the supernatant was discarded. After that the pellet was suspended in the residual fluid and vortexed to suspend evenly. 200 µl of lysis buffer (2 % Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris-HCl), 1mM EDTA, pH 8.0 and 0.2 g of glass beads were added to each Eppendorf tube. Then 200 µl of the solution phenol/chloroform/isoamyl alcohol (25:24:1) was added to the tubes under the fume hood and tubes were placed on rotator and left to mix for 3 min. 200 µl of TE buffer was added and spun for 5 min at maximum speed, the water phase was transferred to new tubes. 1 ml of cold 96 % ethanol was added, mixed and then spun for 5 min at maximum speed at 4°C. the supernatant was discarded and the pellet re-suspended in 400 µl of TE buffer (40 mM Tris-Base, 20 mM acetic acid, 1 mM EDTA, pH 8.0). 6 µl of 7.5 M ammonium acetate was added and the pervious step was repeated. 700 µl of 70 % ethanol was added and spun for 2 min at maximum speed. The supernatant was discarded and dried at 37°C, then finally DNA precipitate was re-suspended in 100 µl of TE …show more content…
225 μl of solution G1 was added to 45 μl DNA in a 1 ml micro-centrifuge tubes and mixed briefly. The mixture was centrifuged at 15 000 g for 1 min at room temperature (Dragon LAB D2012 Centrifuge), then transferred from the micro-centrifuge tubes to collection tubes. The tubes were centrifuged for 60s at 15 000 g. The through flow was discarded. 300 μl of A1 solution was added to the column then centrifuged at 15 000 g for 1 min. The flow through was discarded. 200 μl of A1 solution was added and centrifuged at 15 000 g for 2 min. The column was then placed in a new micro-centrifuge tube. 25 μl of DNA elution buffer was added directly to the column matrix and incubated at room temperature for 3 min. To elute the DNA, the columns were then transferred to 1.5 ml micro-centrifuge tubes and centrifuged at 15 000 g for 2 min. The (quantity and quality of the DNA was tested by spectroscopic measurement (Synergy H4 Hybrid Reader, BioTek) at 260 nm and 280 nm, and the ratio was the criterion for DNA