Edinburgh/7 August 2008


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Thursday 7 August 08

 * Subcultured from Plates 89-90 (BABEL2+glgC-mut1,2,3) to Plate 94. Plate 91 (pSB1A2+glgC-mut1,2) showed no growth, so was discarded. Plate 92 (pSB1A2+glgC-mut1,2) had a blue smear and two white colonies. These colonies were subbed to Plate 95. (HX)
 * Maxiprep X8 (as M67: pSB1A2+rbs+crtB) made. Slight hiccup after step 6. (Cold solution 3 was added and incubation on ice was skipped. Centrifugation continued for 2 minutes before mistake was realised, after which the solution was mixed and incubated on ice for ten minutes (continuing with step 7). (AM, AH)
 * Ligation of rbs+crtB (from M67) and pSB1A2+rbs+crtI (from M50): rbs+crtB as an insert cut with EcoRI/SpeI; rbs+crtI+pSB1A2 as a vector cut with EcoRI/XbaI (L32). (Yan, AM)
 * Double digestions performed: 2 of pSB1A2+rbs+dxs (M72), pSB1A2+rbs+crtE (M63) and pSB1A2+rbs+lims1 (from 0713536 in iGEM 07 box): pSB1A2+rbs+dxs as a vector cut with SpeI/PstI; rbs+crtE as an insert cut with XbaI/PstI; rbs+lims1 as an insert cut with XbaI/PstI.
 * Attempted PCR of cenA (P52) and cex (P53) again using heat killed C. fimi as the template. The PCR products were checked on unnumbered gel, but failed again! (Yan, AM)
 * PCR P54 of PcstA performed. Run on gel 40. Results look promising (fairly prominent band <500bp). Purified. (CF)
 * Plates made for testing glycogen assay. 2x LB agar, ampicillin; 2x LB agar, ampicillin, 2% glucose ready for spreading with E. coli. (SK)
 * Plates 96-97 (glycogen assay) were spread with subcultures from Plate 43 (pSB1A2+rbs+dxs). Plate 96 = (-)glucose, plate 97 = (+)glucose. (HX)


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