Standard Operating Procedure
PCR Protocol
Department: Agronomy
Created by: Chunbin Lu
Laboratory: Crop Production & Physiology
Supervisor: Mark Westgate
Lab Supervisor: Maria Hartt Eckerman
Date approved:
Procedure Overview: This procedure is used for DNA amplification
Equipment and reagents necessary:
Micropipettor and tips (P20, 100)
0.2 ml PCR tubes, 50
Ice bucket and ice
DNA template: 5 ng/ 1 ul
100 ng / 1ul Primer A: AGCAGTTCACCAAGT
100 ng / 1ul Primer B: CGAGCCAGTCAATG
Promega PCR Core system I
Sterile distilled water: 36uL
PCR Thermocycler
Note: Wear gloves and prepare reactions on clean work surface to prevent any
contamination of DNA
Procedure:
1. Using micropipettors, set up PCR reactions in 0.2 mL PCR tubes on ice. Setup of 50
μL reaction includes:
1 uL of DNA Template
1 uL Primer A
1 uL Primer B
5 uL PCR Buffer (Promega PCR Core system I)
5 uL dNTP mix (Promega PCR Core system I)
36 uL sterile dd H2O
2. Add 1 uL Taq Polymerase (Promega PCR Core system I)
3. Place the PCR tubes in PCR Thermocycler.
4. Set the PCR program as per manufacturer’s directions.
The reaction is divided into 3 repeated cycles, 1 minute for each of the 3 steps:
Denaturation at 95°C
Annealing at 65°C
Extension at 72°C
5. There are 30 runs per cycle for each reaction.
Personal Protective Equipment / Engineering Controls:
Skin protection (proper shoes, gloves, lab coat, etc.)
Ventilation system
Hazard controls:
Promega PCR Core system I: US Non Hazardous list
Waste Disposal Procedures & Decontamination:
No waste produced for this procedure.
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