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Edinburgh/12 August 2008
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* Purified P57 (''cex'' from cell suspension) and P64 (''cex'' from genomic DNA). Digested both with PstI. (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) | * 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! | + | * 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) | * 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): | * Analytical digests as follows (AH): | ||
Revision as of 11:39, 20 August 2008
Week 9
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)
