Edinburgh/12 August 2008

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Edinburgh iGEM 2008

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Week 9

Tuesday 12 August 08

Plate 115 made with subs from L33 (Pl. 109/110: pSB1A2+P_cstA), L34 (Pl. 111/112: pSB1A2+dxs+crtE) and L35 (Pl. 113/114: pSB1A2+dxs+lims1) (CF)
Over-night cultures of L33 (pSB1A2+P_cstA, 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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