Edinburgh/Week 17

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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.