Performed 38 more Candida albicans overlays of Bioassays. 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.
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.
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. Colonies one and two from the plate transformed with BL21 (pET28AntA + AntGRFP) cells looked unusual in the soluble and insoluble fractions. They appeared to contain unexpected DNA. But Colonies three and four from the same plate looked promising and would need further investigating to establish whether they contain two plasmids.