IGEM:University of East Anglia (UEA), Norwich, UK/2009/Notebook/NRP-UEA-Norwich iGEM/2013/08/30

iGEM Project name 1

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Floor One Performed 38 more Candida albicans overlays of Bioassays. Fig 1. Gel image showing a 1Kb+ ladder and 8 rows of GUS PCR. All show a band at approximately 1.812Kb. Made up 2 x 200 ml SNA - 200 ml distilled water, 1.6 g nutrient broth powder and 1 g agar. This was autoclaved and left too cool in 55°C water bath (removed before molten stage). A 200 ul volume of Candida was added to 5 ml SNA per plate before swirling to disperse. Agarose gel electrophoresis was performed on the gradient PCR products (110 mV for 35 min). The GUS gene was visible in all reactions at approx. 1.8 Kb (including the new restriction sites) fig 1. Filtered, concentrated and resuspended more spore stocks in glycerol. Performed a QIAquick gel extraction of gel to retrieve the new GUS gene. Re-streaked the conjugations (S4, S4 △antA) onto SFM (+ Ny) with Thiostrepton. Floor Two The ligation reactions from the previous day were retrieved and transformed onto LB agar + Chlorophenicol plates and left at room temperature for 48 hours. Minipreps were performed on the inoculated cultures of samples 3a and Bba_J04450 and left in the -20°C freezer. The pellets of BL21 cells with pET28AntA plasmid and BL21 cells with pET28AntA and AntGRFP plasmids were retrieved. The soluble and insoluble fractions were prepared and analysed on a 15% SDS-PAGE gel.