Double-Distilled Water Lab Report

34. Mixture for overlapping PCR: 1 μL of Herculase II Fusion DNA polymerase, 10 μL of 5X Herculase II reaction buffer, 0.5 μL of a 100mM solution of dNTPs (25 mM each), 2 μL of a 10 μM solution of each primer, 100 ng of each upstream and downstream fragments, 200 ng of pyrG marker fragment and adjust to 50 μL of double-distilled water.
35. Lysis Buffer: to prepare 50 mL of buffer dissolve 23.6 g of Guanidine thiocyanate (118.16 g/L) in 25 mL of double-distilled water. Once dissolved add: 2.5 mL of 1 M Tris-HCl pH 7.0 (121.14 g/L), 2 mL of 0.5 M EDTA pH 8.0 (186.12 g/L) and 0.05 mL of Triton X-100. Add double-distilled water to 50mL (final concentrations: 4 M guanidine thiocyanate, 50 mM Tris-Hcl pH 7.0, 20 mM EDTA and 0.1 % Triton X-100).
36. DNA Binding Solution: First, wash diatomaceous earth (silica particles) by resuspending 5 g in 50 mL of water and allowing them to settle. Decant the supernatant after 30 s and repeat this step twice keeping the supernatant. Centrifuge at 1,400 x g for 1 min, discard the supernatant and resuspend in 25 mL of
Collect fresh spores from mycelium grown in YPGS pH 4.5 for 4-6 days at 26 ºC in the light. Release spores from the sporangia by pouring 10 mL/plate of sterile distillated water on the mycelium and spreading it with a Drigalski spatula. Take the water with the spores by using a pipette and put in a conical tube.
2. Wash the spore solution by centrifugation for 5 min at 440 × g. Resuspend the spores in 10 mL of sterile distillated water and repeat the centrifugation. Resuspend the pellet in 5 mL of sterile distillated water and store the spore solution at 4 ºC. Count the spores using a hemocytometer and light microscope.
3. Take an aliquot of spores containing 2.5 × 108 (no more than 1 week old) and centrifuge for 5 min at 440 × g. Resuspend the pellet in 25 mL of YPG medium pH 4.5 (final spore concentration to 107 spores/mL), supplemented with 200 μg/ml of uridine when a uridine auxotroph is going to be