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Protocol

Quantification of DNA by Real-Time Polymerase Chain

Reaction (PCR)
Michael R. Green and Joseph Sambrook

There are few differences between the experimental steps necessary for amplifying template DNA in a
real-time thermocycler and a standard polymerase chain reaction (PCR). In real-time PCR, it is
necessary, however, to optimize the concentration of primers and probe and to perform a standard
curve. It is also important to consider the data analysis method that will be used.

MATERIALS

It is essential that you consult the appropriate Material Safety Data Sheets and your institution’s Environmental
Health and Safety Office for proper handling of equipment and hazardous material used in this protocol.

RECIPES: Please see the end of this protocol for recipes indicated by <R>. Additional recipes can be found online at
http://cshprotocols.cshlp.org/site/recipes.

Reagents
Forward and reverse primers (optimized concentrations; see Protocol: Optimizing Primer and Probe
Concentrations for Use in Real-Time Polymerase Chain Reaction (PCR) Assays [Green and
Sambrook 2018a])
Nuclease-free water
Probe, optimized concentration (if using TaqMan chemistry; see Protocol: Optimizing Primer and
Probe Concentrations for Use in Real-Time Polymerase Chain Reaction (PCR) Assays [Green
and Sambrook 2018a])
Real-time PCR master mix for SYBR Green I or TaqMan assays <R>
Alternatively, preformulated real-time PCR master mixes containing all of the reagents required for PCR (except
template and primers) in an optimized buffer are available from several vendors (e.g., Thermo Fisher, QIAGEN,
Bio-Rad). These master mixes are designed to simplify experimental setup, decrease the possibility of contami-
nation, and provide optimal performance.

Template DNA
Real-time PCR can only be used reliably when the template can be amplified and when the amplification method
is without error. PCR will only amplify DNA with an intact phosphodiester backbone between the priming sites. In
addition, DNA containing lesions that affect the efficiency of amplification, such as abasic sites and thymine
dimers, will be either underrepresented or may not be represented at all in real-time PCR. Finally, some additives
(e.g., DMSO) and contaminants (e.g., SDS, which can be residual from extraction procedures) can inhibit the
DNA polymerase, thus affecting the results of the real-time PCR. If long oligonucleotides are used as the amplicon
target molecule, they should be PAGE-purified.

Equipment
Barrier tips for automatic micropipettes
Microcentrifuge tubes (0.4–1.5 mL, sterile)

From the Molecular Cloning collection, edited by Michael R. Green and Joseph Sambrook.
© 2018 Cold Spring Harbor Laboratory Press
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot095034

843
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M.R. Green and J. Sambrook

PCR plasticware (e.g., PCR tubes, strips, or 96/384-well plate)

Plasticware should be chosen based on the instructions of the thermal cycler manufacturer. When a block system
is used, the plates must fit well to ensure efficient thermal transfer and uniformity between wells. If the signals are
detected through the cap of the system, optical-grade caps have to be used. To avoid sample evaporation, it is
essential to ensure that the seal of the tubes or plates is complete; some systems—but not all—are compatible
with heat-sealed film coverings that work very well.

Real-time PCR thermal cycler

METHOD

Before running a real-time PCR experiment, it is important to optimize the concentration of the primers (and probe, if
using TaqMan chemistry) and determine the efficiency, sensitivity, and reproducibility of the assay (see Protocol:
Optimizing Primer and Probe Concentrations for Use in Real-Time Polymerase Chain Reaction (PCR) Assays
[Green and Sambrook 2018a]). When designing a real-time PCR experiment, it is important to consider the type of
quantitation (data analysis) method that will be used (see Introduction: Analysis and Normalization of Real-Time
Polymerase Chain Reaction (PCR) Experimental Data [Green and Sambrook 2018b]). If absolute quantification will
be used, a standard curve must be run in parallel with the test samples (see Protocol: Constructing a Standard Curve
for Real-Time Polymerase Chain Reaction (PCR) Experiments [Green and Sambrook 2018c]). The standard curve
may use plasmid DNA or other forms of DNA in which the absolute concentration of each standard is known. One
must be sure, however, that the efficiency of PCR is the same for the standards as for the unknown samples. If either of
the relative methods of quantification will be used, an endogenous reference gene must also be analyzed in parallel
with the test samples.
1. Add 5 µL of the DNA sample(s) to each reaction tube in triplicate. Also include a negative control
(i.e., without template), in which 5 µL of H2O has been added, in triplicate.
2. In a sterile microcentrifuge tube, prepare a PCR mixture according to the tables below, as
appropriate for the type of experiment. The total volume is 15 µL per reaction; multiply each
volume by the number of reactions you need.
When preparing PCR mixtures, it is a good idea to prepare enough mixture for one or two extra reactions to
ensure that all samples and controls have been accounted for, because pipetting inaccuracies can occur.
When a hot-start Taq polymerase such as Applied Biosystems’ AmpliTaq Gold DNA Polymerase (Thermo
Fisher) is used, a 9–12-min pre-PCR heat step at 92˚C–95˚C is required to activate the enzyme. Because
AmpliTaq Gold is inactive at room temperature, there is no need to set up the reaction on ice.
SYBR Green I
Component Volume per reaction (µL)
H2O 3
Forward primer (optimized concentration) 1
Reverse primer (optimized concentration) 1
SYBR Green I Master Mix 10

TaqMan
Component Volume per reaction (µL)
H2O 2.5
Probe (optimized concentration) 0.5
Forward primer (optimized concentration) 1
Reverse primer (optimized concentration) 1
TaqMan Master Mix 10

3. Add 15 µL of the PCR mixture to each sample in the tubes/wells. Mix gently by repeatedly
pipetting up and down, ensuring that no bubbles are produced. Cap the tubes/wells carefully.
Briefly centrifuge the reaction to collect the contents at the bottom of the well or tube.
4. Place the plate or tubes in the real-time thermocycler. Program and run the machine using the
following thermal cycling parameters:
The PCR should be run as soon as possible after preparing the reaction mixes.

844 Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot095034

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Quantification of DNA by Real-Time PCR

SYBR Green I
Temperature (˚C) Time
Initial steps
1. AmpErase UNG activationa 50 2 min
2. AmpliTaq Gold activation 95 10 min
PCR (40 cycles)
3. Melt 95 15 sec
4. Annealing/extension 60 1 min
Dissociation curve
5. Melt 55–95
a
This step is required only when using master mixes containing AmpErase UNG.

TaqMan
Temperature (˚C) Time
Initial steps
1. AmpErase UNG activationa 50 2 min
2. AmpliTaq Gold activation 95 10 min
PCR (40 cycles)
3. Melt 95 15 sec
4. Annealing/extension 60 1 min
a
This step is required only when using master mixes containing AmpErase UNG.
5. Following the PCR run, analyze the raw data (see Introduction: Analysis and Normalization of
Real-Time Polymerase Chain Reaction (PCR) Experimental Data [Green and Sambrook 2018b]).

RECIPE

Real-Time PCR Master Mix for SYBR Green I or TaqMan Assays

dNTPs, 200 µM each (final concentration)
If dUTP is substituted for dTTP (see UNG below), it should be present at a final concentration of
400 µM.

MgCl2, 4–7 mM
Passive reference dye (e.g., 5-carboxy-X-rhodamine [ROX]), 0.45 nM
The fluorescent dye ROX serves as an internal reference when carrying out real-time PCR using
Applied Biosystems instruments. However, its presence does not interfere with reactions performed
using the LightCycler (Roche) or iCycler (Bio-Rad).

Taq polymerase, 0.5 U (0.1 µL)

The specificity of the real-time PCR can be enhanced by using a hot-start Taq polymerase, such as
JumpStart Taq (Sigma-Aldrich), HotStarTaq (QIAGEN), or AmpliTaq Gold (Thermo Fisher). Typ-
ically, 0.1 µL (0.5 U) of Taq DNA polymerase (5.0 U/µL) is added per 20-µL reaction. If necessary,
the reaction can be optimized by increasing the amount by 0.1-U increments. To perform a
TaqMan assay, it is necessary to use a Taq enzyme with 5′ –3′ nuclease activity, such as AmpliTaq
or AmpliTaq Gold (Thermo Fisher).

Uracil-N-glycosylase (UNG; e.g., AmpErase UNG [Thermo Fisher]) (optional)

Products from previous PCR runs are a potential source of contamination in real-time PCR assays. If
contamination from carryover PCR products is suspected, addition of UNG can enzymatically
destroy contaminants and prevent the reamplification of carryover PCR products. In master mixes
containing UNG, dTTP is partially or completely substituted by dUTP. UNG acts by removing uracil
incorporated into any contaminating molecules, and contaminating molecules are destroyed by
cleavage at the apyrimidinic sites generated by the uracil removal. During subsequent cycling,
only target nucleic acid and not contaminating nucleic acid from previous reactions will be amplified.

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M.R. Green and J. Sambrook

REFERENCES

Green MR, Sambrook J. 2018a. Optimizing primer and probe concentra- Green MR, Sambrook J. 2018c. Constructing a standard curve for real-time
tions for use in real-time polymerase chain reaction (PCR) assays. Cold polymerase chain reaction (PCR) experiments. Cold Spring Harb Protoc
Spring Harb Protoc doi: 10.1101/pdb.prot095018. doi: 10.1101/pdb.prot095026.
Green MR, Sambrook J. 2018b. Analysis and normalization of real-time
polymerase chain reaction (PCR) experimental data. Cold Spring
Harb Protoc doi: 10.1101/pdb.top095000.

846 Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot095034

 Downloaded from http://cshprotocols.cshlp.org/ at Stanley Manne Children's Res Inst on October 1, 2018 - Published by
Cold Spring Harbor Laboratory Press

Quantification of DNA by Real-Time Polymerase Chain Reaction (PCR)

Michael R. Green and Joseph Sambrook

Cold Spring Harb Protoc; doi: 10.1101/pdb.prot095034

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