IGEM:University of East Anglia (UEA), Norwich, UK/2009/Notebook/NRP-UEA-Norwich iGEM/2013/08/27

iGEM Project name 1

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Floor One The Bioassay plates had to be overlayed with Candida albicans. Made 3 x 200 ml SNA (soft nutrient agar)- 200 ml distilled water, 1 g agar and 1.6 g broth powder. Autoclaved for 1 hr and then left to cool in 55 °C water bath (being careful not to let it become molten, as this kills Candida). A 200 ul volume of the Candida culture and 5 ml of SNA were added per plate and dispensed by gentle swirling. The Bioassays were then incubated at 30°C. PCR was set up in order to introduce NdeI and SpeI sites at the ends of the GUS gene. The Master Mix contained the following parts and volumes: 32 ul Buffer, 4.8 ul DMSO, 3.2 ul dNTPs, 3.2 ul Template, 4 ul Primer 1 (25 uM), 4 ul Primer 2 (25 uM), 4.8 ul MgCl2, 100.4 ul dH20 and 1.6 ul Phusion enzyme. A total of 8 reactions consisting of 20 ul was made from this. The temperature cycle consisted of: 98°C for 30 sec, 35 cycles of 98°C for 10 sec followed by 72°C for 60 sec and then 72°C for 10 min. Made 1% agarose gel, dissolved the agarose and added 2.5 ul EtBr before pouring.