HOST-DIALOGSOP-HD-001 · v1.1
Day 0 — Bench Cardprint · laminate · BSC-side
Strain: USA300 JE2
Medium: CDM (Hussain)
Volume: 100 mL master · 10 mL × n daughters
PRE-FLIGHT (T−30)
incubator @ 37 °C CDM pre-warmed tracer dilutions fresh drug stocks thawed flasks labelled tubes on ice TCA on ice 90 °C bath ON acid-fix buffer cold scintillation vials lined up timer ready log sheet open
Time Clock Arm Action Volume / spec
T−90 07:30 → 08:00 prep Dilute preculture into master flask.
From overnight (OD ≈ 3) into pre-warmed CDM to OD₆₀₀ = 0.05.
 100 mL · 500 mL flask
T−60 08:30 prep First OD₆₀₀ check. Begin 15-min monitoring.
Expect OD ≈ 0.10–0.15. Record. If far off, flag and continue.
 200 µL aliquot
T−15 09:15 prep Pre-warm daughter flasks. Final label check.
Verify date · strain · drug · arm · replicate on every flask. Confirm drug working dilutions ready.
 n × 50 mL flasks
T 0 09:30 SPLIT + DRUG SPLIT MASTER · APPLY DRUGS SIMULTANEOUSLY.
10 mL master into each daughter via 25 mL serological. Add drug/vehicle within 30 s of split. Start main timer.
 10 mL/flask + drug
T 0 09:30 (master) CLEAN Sample T0 from MASTER (pre-split).
This is the shared baseline for all RNA arms. Pellet, decant, snap freeze.
 1.5 mL × 2 (AU + sRNA)
T 15 09:45 CLEAN Sample T15 per condition.
AU sample → cold acid-fix buffer (0.3 M NaOAc pH 4.5). sRNA sample → TRIzol LS direct on pellet.
 1.5 mL × 2
T 30 10:00 HOT Add ³H tracer to hot daughter flasks.
100 µL tracer working stock per 10 mL → final 0.05 µCi/mL. Record exact addition time per flask. Return to shaker.
 100 µL ³H stock
T 30 10:00 CLEAN Sample T30 per condition.
As T15. Speed matters — pellet to ice within 60 s, freezer within 10 min.
 1.5 mL × 2
T 45 10:15 HOT PULSE END. Quench + TCA-precipitate.
3 × 1 mL aliquots → 1 mL ice-cold 10 % TCA. Mix, ice 20 min.
 3 × 1 mL → TCA
T 45 10:15 CLEAN Sample T45 per condition.
Mid-pulse equivalent on the regulatory side.
 1.5 mL × 2
T 60 10:30 CLEAN Sample T60 per condition.
Post-pulse continuing-effect window.
 1.5 mL × 2
T 45+ 10:35 → HOT Filter onto GF/C · hot-TCA wash.
3 × 5 mL ice-cold 10 % TCA wash on manifold. Then 90 °C in 5 % TCA × 20 min. Cold 5 % TCA wash. 2 × cold EtOH wash. Air-dry.
 GF/C 25 mm
T 120 11:30 CLEAN Sample T120 per condition. Last clean-arm sample.
Late regulatory response window.
 1.5 mL × 2
T ~150 12:00 → HOT Vials + 10 mL Ultima Gold · count.
Tri-Carb, ³H channel (or dual ³H/¹⁴C), 2 min/vial. DPM into log sheet then into shared drive.
 10 mL cocktail
T ~180 13:00 → CLEAN Acid-fix arm: phenol extract · ethanol pp.
Process all AU samples within 4 h of collection. Resuspend in cold acid buffer. Hold at 4 °C max overnight.
 200 µL aq.

Acceptance gates · stop and rerun if any fail

  • Master OD at split: 0.48–0.52
  • Timing deviation per time-point: < 2 min
  • Vehicle hot-arm CV: < 15 %
  • CAM hot-arm reduction: > 70 % all tracers
  • Sample → ice: < 60 s from decant
  • Sample → −80 °C: < 10 min from collection

The 5-second mechanism reminder

  • S. aureus has no AsnRS. Free Asn cannot rescue a GatCAB block.
  • ³H-Asp is the cleaner probe — bypasses the AnsA deamination delay.
  • The diagnostic ratio: [³H-Asn]/[³H-Leu] drops < 0.6 = GatCAB-specific.
  • If all four tracers drop equally = generic translation hit, not the mechanism.
  • Acid-urea: the headline. Asp-tRNA-Asn band rising = direct biochemical proof.
  • Speed kills aminoacyl bonds. Cold + acid + fast, always.
RAD
³H is a pure beta emitter — internal contamination is the hazard, not external dose. Double-glove. Bench paper down. Pipette below surface. Swipe-test hands and bench BEFORE leaving the hot zone. No food, drink, cosmetics anywhere in the radiation area. Spill > 1 MBq → call radiation officer within 24 h.