Standard Operating Procedure
Title: Amplification DNA by PCR
Department: Agronomy
Created by: Haiyan Huang
Laboratory: Crop Production & Physiology Lab Suite
Supervisor: Dr.Jianming
Lab Supervisor: Whitney Bouma
Date approved:
Procedure Overview:
Amplification DNA by PCR
Equipment and reagents necessary:
Equipment: PCR Thermal Cyclers; Micro centrifuge;
Reagent:
Taq DNA polymerase (contains: Standard 10X PCR buffer)
dNTP (DEOXYNUC MIX-8 UMOL OF EACH)
Primer
Deoxyribonucleic acid (DNA)
Miscellaneous:
Micropipettors and tips (10ul, 200ul and 1000ul)
Microcentrifuge Tubes (0.2ml or 0.5ml)
Vertex
Procedure:
Usually 20 to 50 μl total in volume and will include the following:
X μl (0.1 to 1 μg DNA)
10X PCR buffer to give a final concentration of 1X
4 mM dNTP mix to give a final concentration of 0.2 mM
Both the forward and reverse primer added at a final concentration of 0.1 μM to 1 μM of each primer
1 unit/μl Taq polymerase
H2O to bring volume to 20 μl to 50 μl
An example 20 μl reaction
1 μl of dsDNA template (~0.1 μg)
2 μl of 10X buffer
1 μl of 4 mM dNTP mix
1 μl of 10 μM forward primer to a final concentration of 0.5 μM
1 μl of 10 μM reverse primer to a final concentration of 0.5 μM
1 μl of 1 unit/μl Taq polymerase
13 μl of water
Combine the reagents in the 0.5-ml tube or the 0.2-ml PCR tube. Be sure to keep the reagents on ice.
Tap tube gently to mix and spin briefly in micro centrifuge to get all contents to bottom, then place on ice
until ready to load in thermocycler. If thermocycler does not have a heated lid, layer thin film of mineral oil
over mixture to prevent evaporation during cycling.
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revised on 06-20-2013
Personal Protective Equipment / Engineering Cont rols:
Nitrile gloves
Safety glasses
Face shield
Dust mask
Latex gloves
Splash goggles
Lab coat
Fume hood
Neoprene gloves
Vented goggles
Apron
Biosafety cabinet
Insulated gloves
Eye wash station
Safety shower
Respirator
Note: Open-toed and heeled shoes are NOT allowed.
Other Control Measures:
Handling & Storage Precautions:
DNA: Store at -20oC upon receipt in an appropriately labelled container.
Taq polymerase: Store at -20oC upon receipt in an appropriately labelled container.
10X buffer: Store at -20oC upon receipt in an appropriately labelled container.
dNTP: Store at -20oC upon receipt in an appropriately labelled container.
Primer: Store at -20oC upon receipt in an appropriately labelled container.
Waste Disposal Procedures:
Unless EH&S specifically instructs otherwise, all chemical/reagent waste (including excess solutions)
must be placed in an appropriately labeled hazardous waste container for EH&S disposal. Compatible
substances may be combined into one waste container.
Spill/Release Containment and Clean Up/Decontamination Procedures:
Taq polymerase: Absorb spill with inert material (e.g. vermiculite, sand or earth), then place in suitable
container. Avoid runoff into storm sewers and ditches which lead to waterways. Clean
up spills immediately, observing precautions in the Protective Equipment section.
Provide ventilation.
10X buffer: Soak up with an inert absorbent material.
dNTP: Soak up with an inert absorbent material.
Primer: Soak up with an inert absorbent material.
Health & Safety Summary for Required Reagents:
10X buffer:May cause irritation to eyes. May cause irritation to eyes.
There is no MSDS available for Primer, so there should not be any significant health or safety risks associated with
this material.
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revised on 06-20-2013
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Taq polymerase
dNTP
10X buffer
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Incompatibilities
Oxidizing agents
Skin, eyes,
respiratory tract
Primer
 The above summary consists of guidelines for proper handling & disposal of
chemicals used in this procedure. You must read and understand the
contents of the entire MSDS(s) before starting this procedure.
References:
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revised on 06-20-2013