Table of Contents
Bacterial PCR
— Madison Otero 2025/08/12 17:29
Overview
PCR protocol for testing bacteria for RNAi inserts.
Materials
- Baxter water
- 2X bacteria master mix
- bacteria in liquid media
- PCR primers
- ice
- flip top tubes
- PCR tubes (eight well strips)
Procedure
- Gather materials as listed above.
- Thaw 2X master mix and primers in tube rack.
- Vortex 2X master mix and primers once thawed.
- Label all tubes.
- Create master mix: baxter water, 2X master mix, primer 1, primer 2 (recipe below)
- Vortex master mix and place in ice.
- Add 49 uL of master mix to each PCR tube (master mix total without bacteria).
- Add 1 uL of bacteria to its corresponding PCR tube and vortex.
- Add PCR tubes to PCR machine with specified settings (APEX 2X bacterial PCR)
| Step | Temperature | Time |
|---|---|---|
| Initial Denaturation | 95°C | 5 min |
| Denaturation | 95°C | 30s |
| Annealing | 5°C Below Melting | 40s |
| Extension | 72°C | 2 min |
| Hold | 4°C | .∞ |
Master Mix Recipe
| Reagent | Amount Per Rxn |
|---|---|
| Baxter H2O | 22 uL |
| 2X master mix | 25 uL |
| primer 1 | 1 uL |
| primer 2 | 1 uL |
| bacteria | 1 uL |
Multiply master mix by how many reactions you need.
Notes
When calculating master mix, always make 2+ more reactions than you need to account for pipetting error. Make a negative control by including the master mix but not adding any bacteria. Change the annealing temperature based on what is listed on the primer bottle. Change the extension time based on how long the sequence is going to be. When pipetting bacteria from a 5 mL conical tube, ensure that only the tip goes inside the tube. Tilt the tube so the tip stays at the top while the opening of it is submerged in the bacteria media.