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Edinburgh/Week 17
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Week 17 (6~12 October 08)
Tuesday 7 October 08
- More PcstA-xylE assays (see spreadsheet).
- Sequence results: M230 and M232 both seem to be intact ISO2, though there is an extra T in the suffix due to using the wrong version of sob – oops, but not really a problem as the whole suffix is there, just goes TAATAATTACTAGT instead of TAATAATACTAGT. Submit M265 (SU1) for sequencing.
- Subbed rbs+cex/cenA colonies to plate 239.
- Transformed L87 (Edinbrick2+bglX), plates 240, 241
Wednesday 8 October 08
- Glycogen samples to Rabah for Raman spectroscopy.
- Minipreps M270 to M275: three each of rbs+cex, rbs+cenA.
- Mutagenic PCR of SU1, ISO2: P??? and ???.
Thursday 9 October 08
- Digests of M270 and M273 show that M273 (rbs+cenA) looks good but M270 does not. Mutagenic PCRs both failed (gel 218).
- Checked M271, 272 (rbs+cex): both bad (gel 219).
- PCR P105, bglXF and vector reverse primer on L74 to generate a bglX product with EcoRI/PstI ends instead of EcoRI/SpeI in case this works better.
Friday 10 October 08
- Digests to clone P105 (bglX) in Edinbricks 1 and 2, ligations L88, L89.
- PCR P107 = mutagenic PCR on SU1 (M165) with extended denturation and added glycerol: worked! Self-ligation = L90.
Saturday 11 October 08
- PstI digests of other 2 rbs+cenA clones M274, M275. M274 looks good, M275 bad (gel 221).
- Minipreps M276, 277, 278 = last three rbs+cex patches. Digests show M276 looks OK, others wrong.
- Mutagenic PCR P108 on ISO2 (M232), faint but looks OK (also gel 221). Self-ligation = L91.
- Transformed L88, L89 (Edinbrick1/2+bglX) and L90 (SU1 mutation), plates 243-248.
Sunday 12 October 08
- SpeI digests of M274 (rbs+cenA) and M276 (rbs+cex) look OK.
- Digests to repeat insertion of Plac into X13 (crtBI) and X21 (dxs.crtE), ligations L92, L93.
- Possible bglX clones subbed to plate 249. Possible SU1 mutant clones subbed to plate 250.
- Transformed L91 (ISO2 mutation) to plates 251, 252.
