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EN QIAGEN Multiplex PCR Handbook PDF
EN QIAGEN Multiplex PCR Handbook PDF
* Contains 6 mM MgCl2.
Technical Assistance
At QIAGEN we pride ourselves on the quality and availability of our technical support.
Our Technical Service Departments are staffed by experienced scientists with extensive
practical and theoretical expertise in molecular biology and the use of QIAGEN
products. If you have any questions or experience any difficulties regarding the QIAGEN
Multiplex PCR Kit, or QIAGEN products in general, please do not hesitate to contact us.
QIAGEN customers are a major source of information regarding advanced or
specialized uses of our products. This information is helpful to other scientists as well as
to the researchers at QIAGEN. We therefore encourage you to contact us if you have
any suggestions about product performance or new applications and techniques.
For technical assistance and more information, please see our Technical Support center
at www.qiagen.com/goto/TechSupportCenter or call one of the QIAGEN Technical
Service Departments or local distributors (see back cover or visit www.qiagen.com ).
Product Specifications
2x QIAGEN Multiplex PCR Master Mix contains:
HotStarTaq DNA Polymerase HotStarTaq DNA Polymerase is a modified form
of a recombinant 94 kDa DNA polymerase,
originally isolated from Thermus aquaticus, cloned
in E. coli. (Deoxynucleoside-triphosphate: DNA
deoxynucleotidyltransferase, EC 2.7.7.7)
QIAGEN Multiplex PCR Buffer Contains 6 mM MgCl2, pH 8.7 (20C)
dNTP Mix Contains dATP, dCTP, dGTP, dTTP; ultrapure
quality
Q-Solution 5x concentrated
RNase-Free Water Ultrapure quality, PCR-grade
Q-Solution
The QIAGEN Multiplex PCR Kit is provided with Q-Solution, an innovative PCR additive
that facilitates amplification of difficult templates by modifying the melting behavior of
DNA. This unique reagent often enables or improves a suboptimal PCR caused by
templates that have a high degree of secondary structure or that are GC-rich.
Unlike other commonly used PCR additives, such as DMSO, Q-Solution is used at just
one working concentration, which has been optimized for the requirements of
multiplex PCR. It is nontoxic, and does not compromise PCR product purity. For further
information, please read Important Notes, page 14 and the protocol on page 23.
Concentration of normalized
primer stock 50 M (50 pmol/l) 100 M (100 pmol/l)
Each primer 20 l 10 l
TE buffer Variable Variable
Total volume 500 l 500 l
* Allows preparation of a 10x primer mix containing up to 12 primer pairs (50 M stock) or containing up to 25
primer pairs (100 M stock).
Methods of analysis
Primer pairs should be carefully designed. In addition to the sequence of primers, the length of
the generated PCR products should also be taken into account. The sizes of the amplicons must
differ sufficiently in order to be able to distinguish them from one another using agarose or
polyacrylamide gels or capillary electrophoresis systems like the QIAxel system or the Agilent
2100 bioanalyzer system (see Tables 3, 4 and 5). For analysis using high-resolution sequencing
instruments, primers must be different sizes or labeled using different fluorescent dyes.
* When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles.
For more information, please consult the appropriate material safety data sheets (MSDSs), available from the
product supplier.
* Efficient separation of PCR products differing in size by about 20 bp is usually possible using standard
molecular-biologygrade agarose. For separation of fragments that differ in size by less than 20 bp, we
recommend using high-resolution agarose, for example MetaPhor agarose (FMC Bioproducts). For more
information, visit www.cambrex.com .
Table 4. Analysis of Multiplex PCR Products using QIAxcel cartridges on the QIAxel system
Fragment
QIAxcel cartridge Application size range Best resolution
QX DNA High High-resolution 15 bp - 5 kb 3-5 bp for fragments
resolution Cartridge* genotyping 100-500 bp
50 bp for fragments
500 bp - 1 kb
200- 500 bp for
fragments 1-5 kb
QX DNA Screening Fast PCR 15 bp - 5 kb 20-50 bp for fragments
Cartridge screening 100-500 bp
50-100 bp for fragments
500 bp - 1 kb
500 bp for fragments
1-5 kb
QX Large DNA Large and long 15 bp - 10 kb 3-5 bp for fragments
Fragment Cartridge size range 100-500 bp
50 bp for fragments
500 bp - 1 kb
100 bp for fragments
1-5 kb
500 bp for fragments
5-10 kb
* QX DNA High resolution Cartridge is the recommended cartridge for analysis of typical multiplex PCR
products up to 1.5 kb.
Template DNA
Both the quality and quantity of nucleic acid starting template affect PCR, in particular the
sensitivity and efficiency of amplification.*
* For further information see our comprehensive brochure Critical success factors and new technologies
for PCR and RT-PCR. To obtain a copy, visit the QIAGEN web site at www.qiagen.com or call one of the
QIAGEN Technical Service Departments or local distributors (see back cover).
955 bp
99 bp
Figure 1. Efficient 16-plex PCR over a Range of Template Amounts. Multiplex PCRs were performed in duplicate
using the indicated amount of genomic DNA and 16 pairs of primers that amplified fragments of 99, 150, 181,
222, 269, 310, 363, 414, 446, 523, 564, 610, 662, 756, 845, and 955 bp. M: 100 bp ladder.
A M + + B M + +
1307 bp
475 bp 518 bp
231 bp 297 bp
C M + + D M + +
1533 bp
955 bp
1059 bp
662 bp
564 bp
708 bp 446 bp
334 bp
99 bp
advanced applications, such as multiplex reactions with more than 10 products or very
low amounts of template, see Appendix F, page 42.
Procedure
1. Thaw 2x QIAGEN Multiplex PCR Master Mix (if stored at 20C), template DNA,
RNase-free water, and primer mix. Mix the solutions completely before use.
It is important to mix the solutions completely before use to avoid localized
concentrations of salts. Preparing a mixture of all primers avoids pipetting of
individual primers for each experiment, reducing pipetting time and increasing
reproducibility of results (for preparation of primer mix see Table 2, page 9).
2. Prepare a reaction mix according to Table 8.
The reaction mix typically contains all the components required for multiplex PCR
except the template DNA. Prepare a volume of reaction mix 10% greater than that
required for the total number of reactions to be performed. For reaction volumes
less than 50 l, the 1:1 ratio of QIAGEN Multiplex PCR Master Mix to primer mix
and template should be maintained as shown in Table 8.
Note: We strongly recommend starting with an initial Mg2+ concentration of 3 mM
as provided by the 2x QIAGEN Multiplex PCR Master Mix.
Standard Multiplex
Component Volume/reaction Final concentration
PCR Protocol
Reaction mix
2x QIAGEN Multiplex
PCR Master Mix* 25 l 1x
10x primer mix,
2 M each primer (see Table 2) 5 l 0.2 M
RNase-free water Variable
Template DNA
Added at step 4 Variable 1 g DNA/50 l
Total volume 50 l
A final primer concentration of 0.2 M is optimal for most primertemplate systems. However, in some cases
using other primer concentrations (0.10.3 M) may further improve amplification performance.
For volumes less than 50 l, the 1:1 ratio of QIAGEN Multiplex PCR Master Mix to primer mix and template
should be maintained.
Mix gently, for example by pipetting the reaction mix up and down a few times. Due
PCR Protocol
to the hot start, it is not necessary to keep samples on ice during reaction setup.
4. Add template DNA (1 g/50 l reaction) to the individual PCR tubes containing
the reaction mix.
For multiplex RT-PCR, the volume of cDNA added (from the RT reaction) as template
should not exceed 10% of the final PCR volume.
5. When using a thermal cycler with a heated lid, do not use mineral oil. Proceed
directly to step 6. Otherwise, overlay with approximately 50 l mineral oil.
6. Program the thermal cycler according to the manufacturers instructions.
Optional: If a thermal cycler with a temperature gradient function can be used,
determine the optimal annealing temperature by performing a gradient PCR.
7. Place the PCR tubes in the thermal cycler and start the cycling program as outlined
in Table 9, page 19.
Each PCR program must start with an initial heat-activation step at 95C for 15
min to activate HotStarTaq DNA Polymerase.
After amplification, samples can be stored overnight at 28C or at 20C for
long-term storage.
Standard Multiplex
Additional comments
PCR Protocol
Initial activation step: 15 min 95C HotStarTaq DNA Polymerase is
activated by this heating step.
3-step cycling:
Denaturation 30 s 94C
Annealing 90 s 5763C If a gradient PCR cannot be
performed, use 60C as the starting
annealing temperature. If the low-
est Tm* of your primer mixture is
below 60C, use 57C as starting
annealing temperature.
Extension 90 s 72C Optimal for targets up to
approximately 1.5 kb in length.
Number of cycles 3045 The number of cycles is dependent
on the amount of template DNA
and the required sensitivity of your
detection method. See Appendix
C, page 36 for guidelines.
Final extension: 10 min 72C
For targets longer than 1.5 kb, an extension time of 2 min may improve performance.
8. Analyze samples using an appropriate detection system, for example agarose gel
electrophoresis (see Table 3, page 10 for choosing the optimal percentage of
agarose), polyacrylamide gel electrophoresis*, or capillary electrophoresis.
The optimal amount of PCR product required to give a satisfactory signal with your
detection method should be determined individually.
* When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective
goggles. For more information, please consult the appropriate material safety data sheets (MSDSs),
available from the product supplier.
Procedure
1. Thaw the 2x QIAGEN Multiplex PCR Master Mix (if stored at 20C), template
DNA, RNase-free water, and primer mix. Mix the solutions completely before use.
It is important to mix the solutions completely before use to avoid localized
concentrations of salts. Preparing a mixture of all primers avoids pipetting of
individual primers for each experiment, reducing pipetting time and increasing
reproducibility of results (for preparation of primer mix see Table 2, page 9).
Microsatellite
RNase-free water Variable
Protocol
Template DNA
Added at step 4 Variable 1 g DNA/50 l
Total volume 50 l
A final primer concentration of 0.2 M is optimal for most primertemplate systems. However, in some cases
using other primer concentrations (0.10.3 M) may further improve amplification performance.
For volumes less than 50 l, the 1:1 ratio of QIAGEN Multiplex PCR Master Mix to primer mix and template
should be maintained.
Additional comments
Initial activation step: 15 min 95C HotStarTaq DNA Polymerase is
activated by this heating step.
3-step cycling:
Denaturation 30 s 94C
Annealing 90 s 5763C If a gradient PCR cannot be
performed, use 60C as the starting
annealing temperature. If the low-
est Tm* of your primer mixture is
below 60C, use 57C as starting
annealing temperature.
Extension 60 s 72C Optimal for targets up to
approximately 0.5 kb in length.
Number of cycles 2540 The number of cycles is dependent
on the amount of template DNA
and the required sensitivity of your
detection method. See Appendix
C, page 36 for guidelines.
Final extension: 30 min 60C
For targets longer than 0.5 kb, an extension time of 90 s may improve performance.
7. Place the PCR tubes in the thermal cycler and start the cycling program as outlined
in Table 11.
Each PCR program must start with an initial heat-activation step at 95C for 15
min to activate HotStarTaq DNA Polymerase.
After amplification, samples can be stored overnight at 28C or at 20C for
long-term storage.
8. Analyze samples using an appropriate detection system, for example automatic
gel-based DNA sequencers or those based on capillary electrophoresis.
The optimal amount of PCR product required to give a satisfactory signal with your
detection method should be determined individually.
Q-Solution
established annealing temperature in combination with the cycling conditions
Protocol
specified in this protocol.
I Annealing time must be 90 seconds.
I Use equal concentrations (0.2 M) of all primers.
I PCR must start with an activation step of 15 minutes at 95C to activate HotStarTaq
DNA Polymerase (see step 7 of this protocol).
I Optional: If a thermal cycler with a temperature gradient function can be used,
determine the optimal annealing temperature by performing a gradient PCR (see
Appendix C, page 36).
Procedure
1. Thaw the 2x QIAGEN Multiplex PCR Master Mix (if stored at 20C), template DNA,
5x Q-Solution, RNase-free water, and primer mix. Mix the solutions completely
before use.
It is important to mix the solutions completely before use to avoid localized
concentrations of salts. Preparing a mixture of all primers avoids pipetting of
individual primers for each experiment, reducing pipetting time and increasing
reproducibility of results (for preparation of primer mix see Table 2, page 9).
2. Prepare a reaction mix according to Table 12.
The reaction mix typically contains all the components required for multiplex PCR
except the template DNA. Prepare a volume of reaction mix 10% greater than that
required for the total number of reactions to be performed. For reaction volumes
less than 50 l, the 1:1 ratio of QIAGEN Multiplex PCR Master Mix to primer mix,
template, and Q-Solution should be maintained as shown in Table 12.
Note: We strongly recommend starting with an initial Mg2+ concentration of
3 mM as provided by the 2x Multiplex PCR Master Mix.
A final primer concentration of 0.2 M is optimal for most primertemplate systems. However, in some cases
using other primer concentrations (0.10.3 M) may further improve amplification performance.
For volumes less than 50 l, the 1:1 ratio of QIAGEN Multiplex Master Mix to primer mix, template, and
Q-Solution should be maintained.
3. Mix the reaction mix thoroughly and dispense appropriate volumes into PCR tubes
or plates.
Mix gently, for example by pipetting the reaction mix up and down a few times.
Due to the hot start, it is not necessary to keep samples on ice during reaction setup.
4. Add template DNA (1 g/50 l reaction) to the individual PCR tubes containing
the reaction mix.
For multiplex RT-PCR, the volume of cDNA added (from the RT reaction) as template
should not exceed 10% of the final PCR volume.
5. When using a thermal cycler with a heated lid, do not use mineral oil. Proceed
directly to step 6. Otherwise, overlay with approximately 50 l mineral oil.
6. Program the thermal cycler according to the manufacturers instructions.
Optional: If a thermal cycler with a temperature gradient function can be used,
determine the optimal annealing temperature by performing a gradient PCR.
Additional comments
Initial activation step: 15 min 95C HotStarTaq DNA Polymerase is
activated by this heating step.
3-step cycling:
Denaturation 30 s 94C
Annealing 90 s 5763C If a gradient PCR cannot be
performed, use 60C as the
starting annealing temperature. If
the lowest Tm* of your primer
mixture is below 60C, use 57C as
starting annealing temperature.
Q-Solution
Extension 90 s 72C Optimal for targets up to
Protocol
approximately 1.5 kb in length.
Number of cycles 3045 The number of cycles is dependent
on the amount of template DNA
and the required sensitivity of your
detection method. See Appendix
C, page 36 for guidelines.
Final extension: 10 min 72C
For targets longer than 1.5 kb, an extension time of 2 min may improve performance.
7. Place the PCR tubes in the thermal cycler and start the cycling program as outlined
in Table 13.
Each PCR program must start with an initial heat-activation step at 95C for
15 min to activate HotStarTaq DNA Polymerase.
After amplification, samples can be stored overnight at 28C or at 20C for
long-term storage.
8. Analyze samples using an appropriate detection system, for example agarose gel
electrophoresis (see Table 3, page 10 for choosing the optimal percentage of
agarose), polyacrylamide gel electrophoresis*, or capillary electrophoresis.
The optimal amount of PCR product required to give a satisfactory signal with your
detection method should be determined individually.
* When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective
goggles. For more information, please consult the appropriate material safety data sheets (MSDSs),
available from the product supplier.
Little or no product
a) HotStarTaq DNA Poly- Ensure that the cycling program included the HotStarTaq
merase not activated DNA Polymerase activation step (15 min at 95C) as
described in step 7 of the protocols (pages 18, 22,
and 25).
b) Pipetting error or Repeat the PCR. Check the concentrations and storage
missing reagent conditions of reagents, including primers and template
DNA. Mix all solutions before use.
c) Primer concentration Use a concentration of 0.2 M of each primer. For
not optimal amplification of long targets (1.5 kb), a primer
concentration of 0.1 M and extension time of 2 min may
improve results. We do not recommend using primer
concentrations higher than 0.30.4 M, as this may
affect multiplex PCR fidelity. Check concentration of
primer stock solutions. For calculation of primer
concentration, refer to Appendix B, page 34.
d) Insufficient number Increase number of PCR cycles. Refer to Appendix C,
of cycles page 35 for guidelines.
e) PCR cycling conditions Check that the correct cycling conditions were used
not optimal (see Tables 9, 11, and 13 on pages 19, 22, and 25,
respectively). Ensure that an annealing time of 90 s was
used. If possible, perform a gradient PCR to determine
the optimal annealing temperature (see Appendix C,
page 36).
f) PCR cycling conditions Check functionality and specificity of primer pairs in single
not optimal reactions. Ensure that primers of sufficiently high quality
were used. Check for possible degradation of the primers
on a denaturing polyacrylamide gel*. If necessary, make
new dilutions of primer mix from primer stock solutions and
store at 20C in small aliquots. Avoid repeated
freezethaw cycles of the primer mix.
* When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective
goggles. For more information, please consult the appropriate material safety data sheets (MSDSs),
available from the product supplier.
Not all products are detectable, or some products are barely detectable
a) Primers degraded or of- Check functionality and specificity of primer pairs in
low quality single reactions. Ensure that primers of sufficiently high
quality were used. Check for possible degradation of the
primers on a denaturing polyacrylamide gel*. If
necessary, make new dilutions of primer mix from primer
stock solutions and store at 20C in small aliquots. Avoid
repeated freezethaw cycles of the primer mix.
b) Primer concentration Use a primer concentration of 0.2 M. For amplification
not optimal of long targets (1.5 kb), a primer concentration of
0.1 M may improve results. We do not recommend using
primer concentrations higher than 0.30.4 M, as this
may affect multiplex PCR fidelity. Check concentration of
primer stock solutions (see Appendix B, page 34).
c) PCR cycling conditions Check that the correct cycling conditions were used
not optimal (see Tables 9, 11, and 13 on pages 19, 22, and 25,
respectively). Ensure that an annealing time of 90 s was
used. If possible, perform a gradient PCR to determine
the optimal annealing temperature (see Appendix C,
page 36).
d) No final extension step, Ensure that the final extension step was performed as
or final extension step described in Tables 8, 10, and 12 on pages 19, 22,
was not optimal and 25, respectively. When detecting multiplex PCR
products under native conditions, a final extension step
of 15 min at 68C for multiplex systems with more than
10 PCR products, or for PCR products longer than 1.5 kb
may improve results. For microsatellite analysis, a final
extension step of 30 min at 60C should be used.
e) Annealing temperature Check that the correct cycling conditions were used
too high (see Tables 9, 11, and 13 on pages 19, 22, and 25,
respectively). Ensure that an annealing time of 90 s was
used. If possible, perform a gradient PCR to determine
the optimal annealing temperature (see Appendix C,
page 36).
f) GC-rich template or Using the same cycling conditions, repeat the multiplex
template with a high PCR using Q-Solution. Follow the protocol on page 23.
degree of secondary Templates with a very high GC content that do not amplify
structure under these conditions should be combined in a separate
multiplex PCR assay using 1x Q-Solution.
* When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective
goggles. For more information, please consult the appropriate material safety data sheets (MSDSs),
available from the product supplier.
g) Sensitivity not If your assay requires very high sensitivity, the sensitivity
high enough of the multiplex PCR can be further increased by an
extended annealing time of 3 min.
Additional products detectable
a) PCR cycling conditions Check that the correct cycling conditions were used
not optimal (see Tables 9, 11, and 13 on pages 19, 22, and 25,
respectively). Ensure that an annealing time of 90 s was
used. If possible, perform a gradient PCR to determine the
optimal annealing temperature (see Appendix C,
page 36).
b) Too many PCR cycles Too many PCR cycles may increase nonspecific
background. Determine the optimal number of cycles by
decreasing the number of PCR cycles in increments of
3 cycles.
c) Annealing temperature Follow the recommendations given in Appendix A,
too low page 33 to determine the appropriate annealing
temperature for your primers. Increase annealing
temperature in increments of 2C. Ensure that an
annealing time of 90 s was used. If possible, perform a
gradient PCR to determine the optimal annealing
temperature (see Apppendix C, page 36).
d) Mg2+ concentration Use an initial Mg2+ concentration of 3 mM as provide
not optimal by the QIAGEN Multiplex PCR Master Mix. In rare cases,
an increase in Mg2+ concentration may increase product
yield. Perform multiplex PCR with different final
concentrations of Mg2+ by titrating in 0.5 mM steps.
e) Primer concentration Use a primer concentration of 0.2 M. For amplification
not optimal of long targets (1.5 kb), a primer concentration of
0.1 M may improve results. We do not recommend using
primer concentrations higher than 0.30.4 M, as this
may affect multiplex PCR fidelity. Check concentration of
primer stock solutions. For calculation of primer
concentration, refer to Appendix B, page 34.
f) Primer design Review primer design. Refer to Appendix A, page 33,
not optimal for general guidelines on multiplex PCR primer design.
g) Some Primers generate Multiplex primer pairs bind in close proximity to each
more than one specific other, for example during amplification of multiple parts
product of a genomic locus. Additional larger products may be
generated by outside primers (see Appendix E, page 37).
* When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective
goggles. For more information, please consult the appropriate material safety data sheets (MSDSs),
available from the product supplier.
c) No final extension step, Ensure that the final extension step was performed as
or final extension step described in Tables 9, 11, and 13 on pages 19, 22,
was not optimal and 25, respectively. When detecting multiplex PCR
products under native conditions, a final extension step
of 15 min at 68C for multiplex systems with more than
10 PCR products, or for PCR products longer than
1.5 kb may improve results. For microsatellite analysis,
a final extension step of 30 min at 60C should be used.
d) Incomplete renaturation Use a final extension step of 15 min at 68C. We
of PCR products due to recommend this for multiplex systems with more than
either low GC content 10 PCR products, or for PCR products longer than 1.5 kb.
or long length of PCR
products
e) Double-stranded PCR products with a low GC content may melt if
products melt during electrophoresed at high voltages. Reduce the voltage to
electrophoresis prevent the running buffer from overheating.
f) Background using Purify PCR products before loading onto the gel
silver staining (e.g., using the QIAquick Gel Extraction Kit or
MinElute Gel Extraction Kit).
If detecting fluorescently labeled multiplex PCR products under denaturing conditions
(e.g., on an automatic sequencer, or by capillary electrophoresis)
Additional products are observed
a) Loading volume is Loading of large volumes may result in additional
too high peaks. Decrease the loading volume until the
background is decreased to a satisfactory level with
acceptable peak heights (typically peak heights <2000
relative fluorescent units on ABI PRISM 310 or 377
Genetic Analyzer).
b) Faint peaks Amplification of some microsatellite DNA sequences
(stutter peaks) before may lead to artifacts, referred to as stutter peaks, which
main peak are usually one repeat unit shorter than the main peak.
We recommend decreasing the loading volume as
described above. If the length of the faint peak is one base
shorter than the main peak, refer to n1 products
detected below.
c) Sample not completely Denature the samples before loading by heating
denatured to 95C for 3 min.
d) n1 products detected Ensure that the final extension step was performed as
described in Tables 8, 10, and 12 on pages 19, 22,
and 25, respectively. If the final extension step was
correctly performed, decrease the number of cycles
and/or template amount.
e) Differing signal Different fluorescent dyes may give differing signal
intensities from intensities on a particular detection instrument, although
fluorescent dyes comparable amounts of PCR product are generated.
We recommend combining fluorescent dyes for
multiplex PCR according to the instructions of the
detection instruments manufacturer.
Faint peaks or no allele peaks
a) Poor capillary Inject the sample again. Check the syringe O-ring for
electrophoresis injection leakage. Check that the fluorescence detection
(size standard also instrument is functioning correctly.
affected)
b) Poor quality formamide Use high-quality formamide for the analysis of samples
used on the ABI PRISM 310 Genetic Analyzer. The conductivity
of the formamide should be <100 S/cm.
If performing RT-PCR
Little or no product
RT reaction error On average only 1030% of the starting RNA in the
RT reaction is transcribed into cDNA. The volume of the
RT reaction added as template should not exceed 10%
of the final PCR volume.
Additional larger products
Contamination with genomic Additional larger products may result from amplification
DNA of contaminating genomic DNA. Pretreat RNA with
DNase I (e.g., using the QIAGEN RNase-Free DNase
Set, cat no. 79254). Alternatively, use primers located
at splice junctions of the target mRNA to avoid
amplification from genomic DNA.
The probability that a primer has more than one specific binding site within a genome
is significantly lower for longer primers. In addition, longer primers allow annealing at
slightly higher temperatures where Taq DNA polymerase activity is higher.
Annealing temperature
If possible, perform a gradient PCR to determine the optimal annealing temperature (see
page 36). Otherwise, use the recommendations in Table 13.
Sequence
When designing primers for multiplex PCR the following points should be noted:
I Avoid complementarity of 2 or 3 bases at the 3' ends of primer pairs to reduce
primer-dimer formation.
I Avoid mismatches between the 3' end of the primer and the target-template
sequence.
I Avoid runs of 3 or more G and/or C at the 3' end.
* When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles.
For more information, please consult the appropriate material safety data sheets (MSDSs), available from the
product supplier.
Example
Primer length: 24 nucleotides with 6 each of A, C, G, and T bases.
Observed A260 (OD) of a 1 in 100 dilution = 0.283
260 = 0.89 x [(6 x 15,480) + (6 x 7340) + (6 x 11,760) + (6 x 8850)] = 231,916
Concentration = A260 (OD)/260 = 0.283/231,916 = 1.22 x 106 M = 1.22 M
Concentration of primer stock solution = concentration of dilution x dilution factor
= 1.22 M x 100 = 122 M
Creating normalized primer stock solutions for the 10x primer mix
Depending on the level of multiplexing in the reaction, determine whether the
required concentration of the normalized primer stock solution is 50 M or 100 M
(Table 2, page 9).
Example
To create 100 l of a 50 M normalized primer stock solution using the primer from the
example above:
Dilution factor = 50 M/122 M = 0.41
Pipet 0.41 x 100 = 41 l stock solution into a tube and add 59 l TE to give a 50 M
normalized primer stock solution.
* To ensure significance, A260 readings should be greater than 0.15.
Table 15. General Guidelines for Choosing the Number of PCR Cycles
Gradient PCR
Many thermal cyclers have a temperature-gradient function. Using this function, it is
possible to easily determine optimal annealing temperatures by generating a
temperature gradient across the heating block for the annealing step.
If your primers conform to the criteria on page 33, we recommended using a gradient
program that includes a temperature range from 5070C. In order to determine optimal
annealing conditions, prepare 3 identical reactions and place in the block positions that
most closely correspond to annealing temperatures of 57, 60, and 63C.
* When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective
goggles. For more information, please consult the appropriate material safety data sheets (MSDSs),
available from the product supplier.
* When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles.
For more information, please consult the appropriate material safety data sheets (MSDSs), available from the
product supplier.
1050 bp
600 bp
M wt ht hm
1050 bp
600 bp
Figure 2. Detection of Transgenes. Transgenic mice were screened using the QIAGEN Multiplex PCR Kit
and a set of 3 primers specific for the recombination activating gene 2 locus. The primers were designed
to generate PCR products of different sizes so that wild-type mice (wt) could be easily distinguished from
heterozygote mutant mice (ht), or homozygote mutant mice (hm). M: markers.
4
d
an
4
2
on
on
on
M
Ex
Ex
Ex
2219 bp
395 bp
242 bp
Figure 3. Generation of Additional PCR Products in Exon-Specific PCR. Two exons from the human HFE gene
were amplified from human genomic DNA either separately, (Exon 4); (Exon 2), or together (Exon 2 and 4).
The PCR products generated from Exon 4 and Exon 2 were 395 bp and 242 bp respectively. However, in the
multiplex PCR an additional product of 2219 bp is observed, generated by the 2 outside primers. M: markers.
QIAGEN Multiplex PCR Kits for fast and efficient multiplex PCR
QIAGEN Multiplex For 100 x 50 l multiplex reactions: 206143
PCR Kit (100) 2x QIAGEN Multiplex PCR Master Mix
(containing 6 mM MgCl2, 3 x 0.85 ml),
5x Q-Solution (1 x 2.0 ml),
RNase-free water (2 x 1.7 ml)
QIAGEN Multiplex For 1000 x 50 l multiplex reactions: 206145
PCR Kit (1000) 2x QIAGEN Multiplex PCR Master Mix
(containing 6 mM MgCl2, 1 x 25 ml),
5x Q-Solution (1 x 10 ml),
RNase-free water (2 x 20 ml)
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5x Q-Solution, 25 mM MgCl2
DNeasy Tissue Kits for isolation of up to 40 g genomic DNA from
animal tissues and cells, yeast, or bacteria
DNeasy Tissue Kit For 50 DNA preps: 50 DNeasy Spin 69504
(50)* Columns, Proteinase K, Buffers,
Collection Tubes (2 ml)
DNeasy Plant Mini Kits for isolation of total cellular DNA from
plant cells and tissues, or fungi
DNeasy Plant Mini For 50 DNA preps: 50 DNeasy Mini Spin 69104
Kit (50)* Columns, 50 QIAshredder Mini Spin
Columns, RNase A, Buffers,
Collection Tubes (2 ml)
Contains 15 mM MgCl2.
* Larger kit sizes and formats available; see www.qiagen.com .
For up-to-date licensing information and product-specific disclaimers, see the respective
QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals are
available at www.qiagen.com or can be requested from QIAGEN Technical Services
or your local distributor.
Purchase of this product is not accompanied by a license under patents owned by Roche
Molecular Systems, Inc. and F. Hoffmann-La Roche Ltd. No rights to real-time PCR or to
any primers, probes or pathogens are conveyed expressly, by implication or by
estoppel.
Hong Kong I Orders 800 933 965 I Fax 800 930 439 I Technical 800 930 425
Ireland I Orders 1800 555 049 I Fax 1800 555 048 I Technical 1800 555 061