🌡️CELLULAR REAGENT

This protocol describes the preparation of a cell-based reagent from bacterial biomass, suitable for use as a crude lysate in molecular biology applications

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Production of cellular reagents using IPTG

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🧪 Materials

• Bacterial biomass grown on a Petri dish (expressing enzyme of interest)

• 1.5 mL microcentrifuge tubes

• Sterile slide or loop for scraping biomass

• Cold PBS 1X (phosphate-buffered saline)

• Centrifuge (≥4000 rpm, with refrigeration)

• Vortex and pipette

• Spectrophotometer (for OD₆₀₀ measurement)

• PCR tubes

• Water bath or heat block at 60 °C

• Vacuum desiccator with silica beads

• Incubator at 37 °C

• Nuclease-free water

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📋 Protocol Steps

1. Collect the biomass from a Petri dish using a sterile slide or inoculation loop, and transfer it into a 1.5 mL microcentrifuge tube.

2. Add 1 mL of cold PBS 1X, vortex briefly, and centrifuge at 4000 rpm for 15 minutes at 4 °C.

3. Remove the supernatant.

4. Repeat the wash step once more, centrifuge again, and discard the supernatant.

5. Resuspend the pellet in 1 mL of cold PBS 1X. Homogenize the suspension by pipetting, then place the tube on a rotator or shaker for 30 minutes at room temperature.

6. Prepare a 1:100 dilution and measure OD₆₀₀ using a spectrophotometer.

7. Calculate the original OD₆₀₀ of the resuspended biomass and dilute it in PBS 1X to reach OD₆₀₀ = 6.5.

8. Aliquot 31 µL of the diluted biomass into PCR tubes — this volume corresponds to 10 PCR reactions per tube.

9. Incubate the tubes at 60 °C for 10 minutes to eliminate any viable cells.

10. Place the PCR tubes (with caps open) in a vacuum desiccator with silica beads.

11. Incubate the desiccator at 37 °C for 48–72 hours to fully dry the samples.

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💧 Rehydration and Use

• To use the dried reagent, rehydrate with 31 µL of nuclease-free water per tube.

• Use the rehydrated solution directly as a PCR template.

• Store dried tubes in a cool, dry place, preferably sealed and with desiccant.

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