Standard PCR Protocol

HOW TO DO PCR
A standard polymerase chain reaction (PCR) setup consists of four steps:

1. Add required reagents or mastermix and template to PCR tubes.

2. Mix and centrifuge.
*Add mineral oil to prevent evaporation in a thermal cycler without a heated lid.
3. Amplify per thermo cycler and primer parameters.
4. Evaluate amplified DNA by agarose gel electrophoresis followed by ethidium
bromide staining.

REAGENTS: WHAT IS NEEDED FOR PCR?

Reagent Used in PCR Recommended Product

Taq DNA polymerase Select Taq DNA polymerase based upon user preference
table below.)

PCR grade water PCR Reagent Water

Primers diluted to working concentration 10 µM working stocks are sufficient for most assays.

Oligos Custom oligos

DNA to be amplified Provided by researcher

Dedicated pipettes

Thermal cycler With various well block sizes or in multiformat

Sterile filter pipette tips

 Sterile 1.5 mL screw-top microcentrifuge
Corning® microcentrifuge tubes with screw cap
tubes

Choose to fit cycler:

Individual thin-walled 200 µL PCR tubes

PCR tubes or plates

200 µL strip tubes

Multiwell plates and plate seal

Deoxynucleotide mix containing 10 mM each of dATP, d

dNTP mix
*readymixes already include dNTPs

WHAT IS TAQ POLYMERASE?

Taq DNA polymerase is a thermostable enzyme derived from the thermophilic
bacterium Thermus aquaticus. It is commonly used to amplify DNA fragments in
PCR. The enzyme is in a recombinant form, expressed in E. coli. It is able to
withstand repeated heating to 95 °C (as is demanded by the PCR technique)
without significant loss of activity. The enzyme has a molecular weight of
approximately 94 kDa by SDS-PAGE with no detectable endonuclease or
exonuclease activity. It has 5'→3' DNA polymerase activity and 5'→3' exonuclease
activity. Each lot of Taq DNA Polymerase is tested for PCR amplification and
double-stranded sequencing. The enzyme is supplied at 5 units/µL and comes with
an optimized 10x reaction buffer.

Standard Taq DNA Polymerase

Use the table below to select an appropriate mix of Taq DNA polymerase for your
reaction conditions. Choose from clear or red dyed formulations with and without
magnesium chloride (MgCl2) or a pre-prepared readymix or master mix with buffer
and dNTPs.

Containing MgCl2 Separate MgCl2 Readymix

 With red dye for
Clear formulation Clear formulation With red dye for dir
direct load on
without dye without dye load on gels
gels

Taq DNA Polymerase

Taq DNA Polymerase REDTaq® DNA REDTaq® ReadyMix™
from Thermus
from Thermus Polymerase PCR Reaction Mix
aquaticus, without
aquaticus (D1806) (D4309) (R2523)
MgCl2 (D4545)

Unit Definition: One unit incorporates 10 nmol of total deoxyribonucleoside

triphosphates into acid precipitable DNA in 30 minutes at 74 °C.

PROCEDURE: STEPS OF PCR

The optimal conditions for the concentration of Taq DNA polymerase, template
DNA, primers, and MgCl2 will depend on the system being utilized. It may be
necessary to determine the optimal conditions for each individual component. This
is especially true for the Taq DNA polymerase, cycling parameters, and the
MgCl2 concentration. It is recommended the enzyme and the MgCl2 be titrated to
determine the optimal efficiency.

1. Add the reagents to an appropriately sized tube in the order provided in the table.
(Select appropriate table for reaction setup: standard or readymix reagent.) For a
large number of reactions, a mastermix without the template should be set up and
aliquoted into reaction tubes. At the end, template should be added to appropriate
tubes.

Standard PCR Reaction

Amount Component

w µL Water

5 µL 10x PCR Buffer (P2192 or P2317)*

1 µL Deoxynucleotide Mix
 w µL Forward primer
(typically 15-30 bases in length)

x µL Reverse primer
(typically 15-30 bases in length)

0.5 µL Taq DNA Polymerase*

y µL Template DNA (typically 10 ng)

z µL 25 mM MgCl2 (use only with buffer P2317)

50 µL Total reaction volume

*Buy buffer and Taq polymerase together: D1806, D4309 or D4545

Readymix PCR Reaction

Amount Component Fina

25 µL Readymix (R2523 or P4600)

w µL Forward primer 0.1-

(typically 15-30 bases in length)

x µL Reverse primer 0.1-

(typically 15-30 bases in length)

y µL Template DNA (typically 10 ng) 200

z µL Water

50 µL Total reaction volume

2. Mix gently by vortex and briefly centrifuge to collect all components to the
bottom of the tube.

Note: Add 50 µL of mineral oil to the top of each tube to prevent evaporation if
using a thermal cycler without a heated lid.

3. Amplify. The amplification parameters will vary depending on the primers and
the thermal cycler used. It may be necessary to optimize the system for individual
primers, template, and thermal cycler.

Typical Cycling Parameters

25-30 cycles of amplification are recommended.

PCR Step Temperature °C

Denature template 94 °C

Anneal primers 55 °C

Extension 72 °C

4. The amplified DNA can be evaluated by agarose gel electrophoresis and

subsequent ethidium bromide staining.

Note: Mineral oil overlay may be removed by a single chloroform extraction (1:1),
recovering the aqueous phase.

Reagents for Nucleic Acid Electrophoresis

• Agarose (precast gels, powder, etc.)

• Buffer such as MOPS-EDTA-sodium acetate, tris-acetate-EDTA (TAE) or tris-
borate-EDTA (TBE)
• Gel loading solution and sample loading buffer for RNA
• Electrophoresis stain or dye such as ethidium bromide

1.
Cheng S, Fockler C, Barnes WM, Higuchi R. 1994. Effective amplification of long targets from cloned inserts and human
genomic DNA.. Proceedings of the National Academy of Sciences. 91(12):5695-
5699. https://doi.org/10.1073/pnas.91.12.5695

2.
Chou Q. 1992. Minimizing deletion mutagenesis artifact duringTaqDNA polymerase PCR byE.coliSSB. Nucl Acids
Res. 20(16):4371-4371. https://doi.org/10.1093/nar/20.16.4371

3.

Innis MA. 1995. PCR Strategies. New York: Academic Press.

4.

Innis MA. 1990. PCR Protocols: A Guide to Methods and Applications. New York: Academic Press.

5.

Innis MA, Myambo KB, Gelfand DH, Brow MA. 1988. DNA sequencing with Thermus aquaticus DNA polymerase and
direct sequencing of polymerase chain reaction-amplified DNA.. Proceedings of the National Academy of
Sciences. 85(24):9436-9440. https://doi.org/10.1073/pnas.85.24.9436

6.

Newton CR. 1995. PCR: Essential Data. 1. Wiley-Blackwell.

7.

Olive DM, Simsek M, Al-Mufti S. 1989. Polymerase chain reaction assay for detection of human
cytomegalovirus.. 27(6):1238-1242. https://doi.org/10.1128/jcm.27.6.1238-1242.1989

8.

Pääbo S, Gifford JA, Wilson AC. 1988. Mitochondrial DNA sequences from a 7000-year old brain. Nucl Acids
Res. 16(20):9775-9787. https://doi.org/10.1093/nar/16.20.9775

9.

Erlich HA. 1989. PCR technology : principles and applications for DNA amplification. New York: Stockton Press.

10.

Sambrook J, Fritsch E, Maniatis T. 1989. Molecular Cloning: A Laboratory Manual. 1. New York: Cold Spring Harbor
Laboratory Press.

11.

Sarkar G, Kapelner S, Sommer SS. 1990. Formamide can dramatically improve the specificity of PCR. Nucl Acids
Res. 18(24):7465-7465. https://doi.org/10.1093/nar/18.24.7465

12.

Winship PR. 1989. An lmproved method for directly sequencing PCR-amplified material using dimethyl sulphoxide. Nucl
Acids Res. 17(3):1266-1266. https://doi.org/10.1093/nar/17.3.1266

LABEL LICENSE STATEMENT

NOTICE TO PURCHASER: DISCLAIMER OF LICENSE

No license is conveyed with the purchase of this product under any of US Patents
Nos. 5,804,375, 5,994,056, 6,171,785, 6,214,979, 5,538,848, 5,723,591,
5,876,930, 6,030,787, and 6,258,569, and corresponding patents outside the
United States, or any other patents or patent applications, relating to the 5’
Nuclease and dsDNA-Binding Dye Processes. For further information contact the
Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City,
California 94404, USA

Materials

Product No. Description

Z721549 AlumaSeal® 96 filmsize 38 μm, thick aluminum foil sealing film for use with 96 well plates,

Corning® microcentrifuge tubes with screw cap1.5 mL microcentrifuge tube, polypropylen

CLS430909
cap, w/ 0-ring, sterile, natural, 500/cs

D7295 Deoxynucleotide Mix, 10 mMMolecular Biology Reagent

Z374962 PCR microtube and cap stripscapacity 0.2 mL

Z374873 PCR microtubes with attached capscapacity 0.2 mL

Z374903 PCR multiwell plates96 well plate for PCR

Z374911 PCR multiwell platessize 384 wells, polypropylene, skirt, non-sterile

P4600 ReadyMix™ Taq PCR Reaction Mixwith MgCl2

D4309 REDTaq® DNA PolymeraseTaq for routine PCR with inert dye, 10X buffer included

REDTaq® ReadyMix™ PCR Reaction MixComplete PCR reagent with standard Taq DNA Po
R2523
dye

D1806 Taq DNA Polymerase from Thermus aquaticuswith 10× PCR reaction buffer containing Mg

D4545 Taq DNA Polymerase from Thermus aquaticuswith 10× PCR reaction buffer without MgCl

W1754 WaterPCR Reagent