1.Polypropylene 250 ml baffled Erlenmeyer flasks with 0.2 μm filter caps
2.Coccidioides spp. arthroconidia
3.Serological pipettes (10 ml, 25 ml, 50 ml)
4.Disposable hemocytometers (iNCYTO C-Chip NI)
5.Filtered pipette tips (10 μl, 200 μl, 1000 μl)
6.Liquid waste container (bottle with screw cap)
7.50 ml polycarbonate, screw cap, conical centrifuge tubes
8.Empty tip box (for use as 50 ml tube holder during horizontal incubation with shaking)
9.15 ml polypropylene, screw cap, conical centrifuge tubes
10.Cold beads in small ice bucket (-20°C)
11.Inoculating loops (1 µl, 10 µl)
12.1.5 ml microcentrifuge tubes
Note: Prepare all media and buffers using ultrapure water (resistivity of 18 M Ω cm at 25°C). Use only molecular biology grade reagents. Store all reagents at 4°C unless otherwise indicated.
A.1% glucose, 0.5% yeast extract
B.Two 100 ml bottles/transformation experiment
2. Dry enzyme powder for cell wall digestion enzyme mixture prepared in a 50 ml (polypropylene) conical centrifuge tube
A.75 mg Driselase fromBasidiomycetes(Sigma), 40 mg lysing enzymes fromTrichoderma harzianum(Sigma), 10 units chitinase (optional) fromStreptomyces griseus(Sigma)
i.The use of chitinase is optional, but it appears to enhance the transformation competency of the protoplasts.
A.50 mM potassium citrate, 0.6 M KCl (pH 5.8)
B.200 ml/transformation experiment
A.10 mM sodium phosphate, 1.2 M MgSO4(pH 5.8)
B.100 ml/transformation experiment
A.100 mM MOPS, 0.6 M sorbitol (pH 7.5)
B.100 ml/transformation experiment
6. MOPS buffer containing sorbitol (MS)
A.10 mM MOPS, 1 M sorbitol (pH 6.5)
B.500 ml/transformation experiment
B.100 ml/transformation experiment