Edinburgh/12 August 2008


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Tuesday 12 August 08

 * Plate 115 made with subs from L33 (Pl. 109/110: pSB1A2+PcstA), L34 (Pl. 111/112: pSB1A2+dxs+crtE) and L35 (Pl. 113/114: pSB1A2+dxs+lims1) (CF)
 * Over-night cultures of L33 (pSB1A2+PcstA, sub-cloned on Plate 115), L34 (pSB1A2+dxs+crtE, sub-cloned on Plate 115) and L35 (pSB1A2+dxs+lims1, subcloned on Plate 115) set up, ready for minipreps tomorrow.
 * M121-M123: Minipreps of pSB1A2+crtY (1,2,3). M124-M126: Minipreps of pSB1A2+rbs+crtY (1,2,3). note: I messed up M121 by adding 200µl instead of 100µl of sol 1. They are in the green box, the orange one is full. (OG)
 * Double digests of M121 to M126, run on Gel 46. It looks good! M121 is good! (Yan)
 * Purified P61 (rrnB) for sequencing. (CF/AM)
 * Re-purified already purified genomic DNA from C. fimi. This meaningless endeavour resulted from a misunderstanding. Hopefully, the doubly-purified DNA will still be useable. (AM)
 * P57 (cex from cells) EcoRI/PstI double digest, undigested P64 (cex from genomic DNA), P66 (su1) and P67 (iso2) run on Gel 45. P57 double digest yielded no clear bands; P64 yielded a clear band at 1.5 kb. (CF/AM)
 * Purified P57 (cex from cell suspension) and P64 (cex from genomic DNA). Digested both with PstI. (AM)
 * PCR for cenA from C. fimi genomic DNA (P65): denaturing 1 min, annealing 56°C for 20s, extension 30s, with glycerol. (AM)
 * P57 and P64 (cex) PstI digests and P65 (cenA) run on Gel 47. Results indicate success! (AM)
 * Re-did the excise of crtE from BABEL2 (M79); cut out the band responsible for crtE and Edinbrick 1 on the gel, followed by putification of crtE and Edinbrick1 from the cut-out gel. Finally, ligation of crtE to Edinbrick1 (L38). (HX, AH)
 * Analytical digests as follows (AH):
 * M109 and M110 (pSB1A2+crtB+crtI): BamHI/EcoRV
 * M115 and M116 (pSB1A2+glgC-mut1,2): HindIII
 * M115 and M116 (pSB1A2+glgC-mut1,2): EcoRI/HindIII
 * Digests of M109, M110, M115 and M116 were run on gel 48. M109 and M110 should give two bands if both genes are present. HindIII digest of M115/116 should give a single band indicating linearised plasmid with glgC. Double digest of M115/116 should give an insert band of ~1kb and a vector + ~500bp of insert band. Results do not look good. Bands seem to indicate much too large products, but will discuss with Chris tomorrow.(AH)
 * M116 (pSB1A2+glgC-mut1,2) and M120 (BABEL2+glgC-mut1,2,3) submitted for sequencing. (CF)


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