Regular PCR
Bench manual and fillable template for routine endpoint PCR setup, thermocycler programming, gel verification, and image recordkeeping.
Before Starting
Use this as a template
Enter the assay-specific annealing temperature, cycle count, template volume, and primer/master mix volumes before preparing reactions.
PCR contamination control
Keep template DNA separate from clean PCR reagents. Use filter tips, change gloves often, and include a no-template control when possible.
Gel check
Record the gel percentage, run settings, ladder, sample loading volume, and final gel image filename in the bench record.
Materials
PCR Reagents
- PCR master mix
- Forward primer
- Reverse primer
- Template DNA
- DI water or nuclease-free water
- No-template control water
Gel Verification
- Agarose gel
- Loading dye, if needed
- DNA ladder appropriate for expected product size
Equipment and Consumables
- PCR tubes, strips, or plate
- Aerosol-resistant tips
- Ice bucket or cold block
- Mini centrifuge or plate spinner
- Thermal cycler
- Gel electrophoresis system
Reaction Calculator
Include samples, controls, and extra reactions. Totals also show 10% overage.
| Component | Per reaction | Total | Total + 10% |
|---|---|---|---|
| PCR master mix | 12.5 µL | 12.5 µL | 13.8 µL |
| DI water | 10.5 µL | 10.5 µL | 11.6 µL |
| Forward + reverse primer mix | 1 µL | 1 µL | 1.1 µL |
| Template DNA | 1 µL | 1 µL | 1.1 µL |
| Final reaction | 25 µL | 25 µL | 27.5 µL |
Check water volume
If the calculated water volume is 0 µL or negative, reduce another component or increase the final reaction volume before setting up PCR.
Part A: Prepare PCR Reactions
~20-40 min
- Thaw PCR master mix, primers, water, and template DNA on ice or a cold block.
- Mix each reagent gently and briefly spin down.
- Label PCR tubes, strips, or plate positions before adding reagents.
- Prepare a master mix containing water, PCR master mix, and primers. Keep template DNA separate until the final addition.
- Aliquot master mix into each reaction tube or well.
- Add template DNA to sample reactions.
- Add water instead of template DNA to the no-template control.
- Seal reactions, mix gently, and briefly spin down before loading the thermal cycler.
Part B: PCR Program
~1.5-2 h, depending on cycle count
| Step | Description | Temperature | Duration |
|---|---|---|---|
| 1 | Initial denaturation | 95°C | 5:00 min |
| 2 | Denaturation | 95°C | 0:30 min |
| 3 | Annealing | 55°C | 0:30 min |
| 4 | Extension | 72°C | 1:00 min |
| 2-4 | Repeat cycling steps | 35 cycles | |
| 5 | Final extension | 72°C | 5:00 min |
| 6 | Final hold / storage | 12°C | Hold |
- Confirm the program name matches the primer set or assay.
- Enter the assay-specific annealing temperature.
- Enter the cycle count.
- Confirm final extension and hold settings before starting the run.
Part C: Labeling and Gel Verification
~30-60 min
- Record sample IDs, primer pair, template source, and plate/tube positions.
- Prepare the agarose gel according to the expected product size.
- Record gel percentage, cooling time, voltage, current if displayed, and run time.
- Load ladder and PCR products according to the gel plan.
- Image the gel and record the image filename.
| Gel record item | Value |
|---|---|
| Gel percentage | |
| Cooling time | 20 min |
| Voltage | |
| Current | |
| Run time | |
| Ladder | |
| Sample loading volume | |
| Gel image filename |
Bench Record
| Date | |
|---|---|
| Operator | |
| Project / assay | |
| Primer pair | |
| Sample IDs | |
| Reaction volume | 25 µL |
| Template volume | 1 µL |
| Annealing temperature | 55°C |
| Cycle count | 35 |
| No-template control | Included / Not included |
| Gel percentage | |
| Ladder | |
| Gel image file | |
| Result summary | |
| Notes |
Bench Notes
Stop Points
Stop and ask before:
- Running PCR if the primer pair, annealing temperature, or expected product size is unknown.
- Continuing if the no-template control amplifies.
- Interpreting a gel if the ladder failed or the gel image filename cannot be matched to the run.