Contributors: Tank
Primer arrival issue
Primers arrived (End-REV-1, Start-FOR-H, Start-REV-1, Mid-FOR-1, Mid-REV-1, End-FOR-1), but Start-FOR-1 was missing — Start-FOR-H had been ordered as a substitute for Mid-FOR-H, while Mid-FOR-H itself was still separately ordered. Net effect: the primer needed for the whole-gene PCR isn’t available, so that PCR cannot proceed this round. End-FOR-1, Start-FOR-H, and Start-REV-1 were rehydrated; the rest were left in the freezer.
Redid the faithful PCR for backbones 2 and 4 with what was available (22.5 µl master mix + 1.25 µl each of the relevant primer pair per tube).
M9 plate dilution-factor correction
Previous control plates were found to have a 0.77 dilution factor (actual volume added vs the intended 320 µl → M9 total, ~250/320 = 0.77) — meaning every stated concentration in those plates was effectively 0.77× the intended value (e.g. “1%” cellobiose was really ~0.77%).
Recalculated stock concentrations to push the dilution factor closer to 1 (i.e. more concentrated stocks, less volume added per plate): glucose 20% w/v, cellobiose 10% w/v, chloramphenicol 34 mg/mL. Reworked volumes per 250 mL M9 batch:
| Target condition | Stock volume needed | Resulting actual concentration |
|---|---|---|
| 0.4% glucose | 5 mL of 20% glucose | 0.392% (with 40 µg/mL chloramphenicol → 39.2 µg/mL) |
| 1% cellobiose | 25 mL of 10% cellobiose | 0.91% (with 40 µg/mL chloramphenicol → 36 µg/mL) |
| 1% cellobiose + 0.4% glucose | both above | 0.356% glucose equivalent (40 µg/mL chloramphenicol → 35.6 µg/mL) |
0.1% w/v yeast extract was added directly to the M9 + agar base for each condition, with the exact mass (0.255–0.280 g) back-calculated against the corrected final volumes so its dilution factor also comes out to 1.