> For the complete documentation index, see [llms.txt](https://reclone-latam-uncuyo.gitbook.io/building-a-reagent-collaborative-network-in-latam/llms.txt). Markdown versions of documentation pages are available by appending `.md` to page URLs; this page is available as [Markdown](https://reclone-latam-uncuyo.gitbook.io/building-a-reagent-collaborative-network-in-latam/cellular-reagent.md).

# 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

***

[Production of cellular reagents using IPTG](https://www.protocols.io/view/production-of-cellular-reagents-using-iptg-4r3l2owzjv1y/v1)

***

### <mark style="color:red;">🧪 Materials</mark>

* 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

***

### <mark style="color:red;">📋 Protocol Steps</mark>

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.

***

### <mark style="color:red;">💧 Rehydration and Use</mark>

* 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.

***