IGEM:University of East Anglia (UEA), Norwich, UK/2009/Notebook/NRP-UEA-Norwich iGEM/2013/08/05
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A total of 49 liquid cultures from streak-purified single colony Streptomyces plates were prepared previously through inoculation in LB broth with springs.
Made up 30 mg/ml lysozyme stock. The mix was filter sterilised and 10 ul added to each eppendorf of liquid culture (49). The liquid cultures were then centrifuged to remove as much supernatant as possible. A 400 ul aliquot of Buffer 1 (TE buffer containing 10mM Tris and 1mM EDTA) was added to each culture containing the lysozyme. The eppendorfs were incubated at 37 °C for 1 hr to allow the lysis reaction to proceed.
PCR was then performed on the 49 lysed cultures. A Master Mix was prepared - 110 ul Buffer, 55 ul DMSO, 22 ul dNTPs, 55 ul Primer 1, 55 ul Primer 2, 33 ul MgCl2, 742.5 ul dH20, 11 ul Taq polymerase. 19.6 ul of Master Mix was added to 0.4 ul of lysed culture (template DNA). The mixes were placed in a PCR machine for a 3 hr cycle: 96°C for 12 mins, (96°C for 1 min, 45°C for 0.45mins, 72°C for 1.3mins) x29, 72°C for 20mins.
Made 4 x 500 ml SFM agar media, autoclaved for 1 hr. Added 5 ml MgCl2 to one flask, 500 ul Nystatin to 3 flasks and poured into plates.