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.