OpenTaq Cellular Reagent Production (Evaporative Drying)

• PC1 (OGTR) / CL1 (HSE) containment required. This procedure produces and manipulates a genetically modified organism (E. coli BL21(DE3) carrying the pATetO-Taq expression plasmid) and cannot legally be performed in a non-certified facility. Australian operators: OGTR-certified PC1 facility. UK operators: HSE-notified CL1 premises under GMO (Contained Use) Regulations 2014.
• BL21(DE3) safety profile. BSL-1 host; non-pathogenic; standard lab-safety precautions apply. Autoclave all liquid and solid waste before disposal.
• Ampicillin. Handle with gloves; avoid skin contact. Waste containing ampicillin should be autoclaved and disposed of per local biosafety waste protocols.
• Anhydrotetracycline (aTC). Chemical inducer; low acute toxicity but handle with standard gloves/eye protection. Dispose per chemical-waste pathway, not down the drain.
• Desiccant (calcium sulfate). Mild skin/eye irritant in dust form. Handle in a ventilated area; avoid inhalation. Consult SDS.
• Heat inactivation prior to end-user distribution. After evaporative drying, heat-treat the sealed aliquots at 60°C for 10 minutes to inactivate any residual viable cells. This is required before any release of the reagent outside the producing facility, even in RUO contexts, because the enzyme is functional after heat treatment but the GMO host is not viable.
• No clinical application. This reagent is for research and educational use only (RUO). Not validated or approved for clinical diagnostic, therapeutic, or regulatory-grade use.

Plasmid + strain. Obtain pATetO-Taq from Addgene (search the Open Enzyme Collection) under OpenMTA terms. Obtain E. coli BL21(DE3) competent cells from NEB, Novagen, or prepare in-house by the standard calcium-chloride or TSS method. Transform and store single-colony glycerol stocks at −80°C before beginning the production run.

Media prep. Superior Broth (Athena Enzyme Systems product; in-house substitute: 32 g/L tryptone, 20 g/L yeast extract, 5 g/L NaCl, autoclaved). Ampicillin stock at 100 mg/mL in water, filter-sterilised, stored at −20°C. Anhydrotetracycline (aTC) stock at 1 mg/mL in 50% ethanol, stored at −20°C protected from light.

Desiccant preparation. Calcium sulfate pellets (or silica gel as substitute); if previously used, regenerate by baking at 200°C for 2 hours and cool in sealed container before reuse.

Bench prep. Dedicated pre-transformation and post-transformation areas. Autoclave flasks, tips, and waste. Keep glove changes strict between handling live culture and dried-reagent aliquots (once aliquots are sealed, treat them as finished product, not live culture).

LibreBiotech setup. Before starting production: create a Process in your production Study referencing this procedure version. Each production batch should be a discrete Process with its own Sample records (one Sample per aliquot strip), linked to the same parent Process. Lot number format recommended: OT-YYYYMMDD-NN (OpenTaq / date / batch-of-day).

• Day 0 (30 min active, then overnight): Plate transformation → 37°C overnight growth.
• Day 1 (15 min active, then overnight): Pick colony into starter culture → 37°C 225 rpm overnight.
• Day 2 (~5 h active): Dilute 1:200 → grow to OD600 0.4–0.7 (~2 h) → induce with aTC (3 h at 37°C) → harvest, wash, aliquot (~30 min).
• Day 2 → Day 3/4 (hands-off): Evaporation at 37°C in desiccant jars, 1–2 days until visibly dry.
• Day 4 (~15 min active): Seal caps, heat-inactivate (60°C × 10 min), label, QC-activity test (small subset), store.
• Total active time per batch: ~6 hours spread across 4 days. Hands-off time dominates.
• Time-sensitive: Do not leave induced cultures at room temperature; proceed from harvest to aliquot within 30 minutes. Do not interrupt the evaporation step once started — reopening the jar mid-drying risks rehydration from ambient humidity.