Download Manual: FullVelocity(TM) SYBR® Green QPCR - Gene X

FullVelocity™ SYBR® Green QPCR
Master Mix
INSTRUCTION MANUAL
Catalog #600581 (single kit)
Catalog #929581 (10-pack kit)
Revision #056003a
For In Vitro Use Only
*600581-12_056003a/*
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FullVelocity™ SYBR® Green QPCR Master Mix
CONTENTS
Materials Provided.............................................................................................................................. 1
Storage Conditions .............................................................................................................................. 1
Additional Materials Required .......................................................................................................... 1
Notices to Purchaser ........................................................................................................................... 1
Introduction......................................................................................................................................... 2
Features of Kit Components.................................................................................................. 2
Passive Reference Dye .......................................................................................................... 3
SYBR® Green I Dye.............................................................................................................. 3
Faster QPCR using the FullVelocity™ SYBR® Green QPCR Master Mix .......................... 3
Fluorescence Monitoring in Real-Time................................................................................. 4
Preprotocol Considerations................................................................................................................ 6
cDNA Synthesis and RNA Isolation ..................................................................................... 6
Primer Design and Concentration ......................................................................................... 6
Reference Dye ....................................................................................................................... 7
Magnesium Chloride Concentration...................................................................................... 7
Preparing a Single Reagent Mixture for Multiple Samples................................................... 7
Mixing and Pipetting Enzyme Solutions ............................................................................... 7
Recommended Control Reactions ......................................................................................... 8
Preventing Template Cross-Contamination........................................................................... 8
Fluorescence Detection and Data Acquisition....................................................................... 8
Protocols............................................................................................................................................... 9
Preparing the Reactions......................................................................................................... 9
PCR Cycling Program ......................................................................................................... 10
Dissociation Program .......................................................................................................... 11
Troubleshooting ................................................................................................................................ 12
References .......................................................................................................................................... 13
Endnotes............................................................................................................................................. 13
MSDS Information............................................................................................................................ 13
Quick-Reference Protocol ................................................................................................................ 15
FullVelocity™ SYBR® Green QPCR Master Mix
MATERIALS PROVIDED
Catalog #600581 (single kit) and Catalog #929581 (10-pack kit)
Materials provided
Quantitya, b
2× FullVelocity™ SYBR® Green QPCR Master Mix
2 × 2.5 ml
Reference dye, 1 mM
a
b
100 μl
Sufficient PCR reagents are provided for four hundred, 25-μl reactions.
Catalog #929581 contains 10 Catalog #600581 kits.
STORAGE CONDITIONS
All Components: Upon receipt, store all components at –20°C. Store the 2× master mix at 4°C after
thawing. Once thawed, full activity of the master mix is guaranteed for 6 months.
Note
The reference dye and the 2× FullVelocity™ SYBR® Green QPCR master mix are lightsensitive and should be kept away from light whenever possible.
ADDITIONAL MATERIALS REQUIRED
Spectrofluorometric thermal cycler
Nuclease-free PCR-grade water
NOTICES TO PURCHASER
Limited Label License
No rights are granted to the purchaser hereunder to sell, modify for resale or otherwise transfer this
product. Stratagene reserves all other rights, and this product may not be used in any manner other
than as provided herein.
Other Notices
Use of certain labeling reagents may require licenses from entities other than Stratagene.
Revision #056003a
FullVelocity™ SYBR® Green QPCR Master Mix
Copyright © 2006 by Stratagene.
1
INTRODUCTION
®
The FullVelocity™ SYBR Green QPCR master mix kit provides rapid,
economical real-time quantification of DNA and cDNA targets.
FullVelocity technology is powered by a novel archaeal DNA polymerase
specifically engineered for high speed amplification. Forty-cycle run times
are reduced by up to 40% compared to conventional QPCR reagents,
depending on the QPCR instrument platform used. The FullVelocity SYBR
Green QPCR master mix includes a thermostable accessory protein that
destabilizes mis-annealed primers at each cycle and neutralizing hot-start
monoclonal antibody that prevents extension of mis-annealed primers
during room temperature reaction set-up. Together, these specificity
enhancers reduce interference from primer-dimers and spurious
amplification products. The accelerated run times, reliability, specificity,
and master mix format make the FullVelocity system an ideal choice for
high-throughput QPCR applications.
The FullVelocity SYBR Green QPCR master mix is easy to use and
compatible with most existing primer-template systems, although some
primer redesign may be necessary to optimize specificity or amplicon size
(90–300 bp amplicons are recommended). The kit has been successfully
used to amplify and detect a variety of high-, medium- and low-abundance
targets from genomic DNA, plasmid DNA, and cDNA. The FullVelocity
reagents and protocols have been optimized for maximum performance on
Stratagene’s Mx4000®, Mx3000P®, and Mx3005P™ instruments, and may
also be adapted for use on most other QPCR platforms.
Features of Kit Components
The 2× FullVelocity SYBR Green QPCR master mix includes a novel
archaeal DNA polymerase specifically engineered for high-speed QPCR.
This DNA polymerase exhibits a number of superior features including
extreme thermostability, lack of 3´-5´ and 5´-3´ nuclease activities, and a
specifically engineered DNA binding domain. In addition to the DNA
polymerase, the 2× FullVelocity master mix includes an optimized PCR
buffer, SYBR green I dye, MgCl2, nucleotides (GAUC), stabilizers,
neutralizing hot start monoclonal antibodies, and a thermostable accessory
protein that destabilizes mis-annealed primers and minimizes interference
from non-specific amplification products. Stratagene’s expertise with
archaeal enzymes has been used to produce a unique enzyme formulation
that supports shorter denaturation and annealing/extension steps and
tolerates temperature changes better than Taq DNA polymerase.
2
FullVelocity™ SYBR® Green QPCR Master Mix
Passive Reference Dye
The passive reference dye (with excitation and emission wavelengths of
584 nm and 612 nm, respectively) is provided as an optional reagent that
may be added to compensate for non-PCR related variations in fluorescence.
Providing the reference dye in a separate tube makes the FullVelocity SYBR
Green QRT-PCR master mix adaptable for many real-time QPCR platforms
(see Reference Dye in Preprotocol Considerations for more information).
SYBR® Green I Dye
The SYBR Green I dye1 has a high binding affinity to the minor groove of
double-stranded DNA (dsDNA). It has an excitation maximum at 497 nm
and an emission maximum at 520 nm. In the unbound state the dye exhibits
little fluorescence; however, when bound to dsDNA, the fluorescence
greatly increases, making it useful for the detection of product accumulation
during real-time PCR. During denaturation, all DNA becomes singlestranded. At this stage, SYBR Green is free in solution and produces little
fluorescence. During the annealing/extension step, the primers hybridize to
the target sequence and are extended, resulting in dsDNA to which SYBR
Green I can bind. The increase in fluorescence signal intensity over
40 amplification cycles depends on the initial concentration of target present
in the PCR reaction.
An important consideration when using SYBR Green I, however, is that it
can bind to nonspecific dsDNA (e.g. primer-dimers and spurious PCR
products) and fluoresce. Therefore, in the early stages of assay optimization,
it is recommended that the PCR products are analyzed on a gel to verify
production of the specific product of interest and to verify a correlation
between the gel appearance and the fluorescence data. With every
experiment, no-template controls should be performed to test for reagent
contamination and primer-dimer formation. In addition, dissociation curve
analysis is essential for discriminating between the synthesis of intended
PCR product and the formation of primer-dimers or spurious amplification
products. In general, careful primer design, optimizing primer concentration,
and using primers of higher purity (e.g. HPLC-purified primers) can
minimize the occurrence of side-reaction products, leading to more reliable
DNA quantification.
Faster QPCR using the FullVelocity™ SYBR® Green QPCR Master Mix
The FullVelocity SYBR Green master mix has been specifically optimized
for use with faster cycling times. For optimal sensitivity and specificity, it is
critical that researchers modify their existing cycling regimen to shorten
denaturation and annealing/extension times. See the PCR Cycling Program
section of Protocols for the recommended cycling conditions for the
FullVelocity SYBR master mix on various QPCR platforms.
FullVelocity™ SYBR® Green QPCR Master Mix
3
Fluorescence Monitoring in Real-Time
When fluorescence signal from a PCR reaction is monitored in real-time, the
results can be displayed as an amplification plot (Figure 1), which reflects
the change in fluorescence during cycling. This information can be used to
quantitate initial copy number based on threshold cycle (Ct).2 Ct is defined
as the cycle at which fluorescence is statistically significant above
background. The threshold cycle is inversely proportional to the log of the
initial copy number.2 The more template that is initially present, the fewer
cycles required to reach the point where the fluorescence signal is detectable
above background. Quantitative information based on threshold cycle is
more accurate than information based on endpoint determinations because
Ct is based on measurements taken during the exponential phase of PCR
amplification when the PCR efficiency is not yet influenced by limiting
reagents and small differences in reaction components or cycling conditions.
Quantitative assessments based on endpoint fluorescence values (a single
reading taken at the end of the PCR reaction) are inherently inaccurate
because endpoint values can be greatly influenced by these factors.
Figure 1 shows Mx3000P instrument amplification and dissociation curve
plots obtained from amplifying a 550 bp amplicon in the absence
(–template) or presence (+template) of template DNA. The reaction
containing template shows a significant increase in fluorescence and has a
Ct value ≅ 23. The reaction without template has a Ct of ≅37. To determine
if the fluorescence in the no-template control reaction arises from true
amplification of contaminating template in the reaction (primer stocks,
master mix) or from the synthesis of primer-dimers or other non-specific
products, a dissociation profile is generated. In the dissociation curve, PCR
samples are subjected to a stepwise increase in temperature from 55°C to
95°C and fluorescence measurements are taken at every temperature
increment. The melting of products causes SYBR Green dissociation,
resulting in decreased fluorescence. After completion of the dissociation
segment, fluorescence is plotted versus temperature. To simplify
interpretation, the first derivative [–R´(T) or –Rn´(T)] should be plotted. As
the temperature increases, the amplification products melt according to their
composition. If primer-dimer or nonspecific products were made during the
amplification step, they will generally melt at a lower temperature (Tm) than
the desired products. The dissociation curve plot of these samples shows
two fluorescence peaks: one in the “– template” reaction centered around
79°C (which corresponds to primer-dimer); and the other, in the “+
template” reaction, centered around 87°C (which corresponds to amplicon).
In this way, the dissociation curve analysis of PCR products amplified in the
presence of SYBR Green I dye can be a very powerful tool in the
interpretation of fluorescence data.
4
FullVelocity™ SYBR® Green QPCR Master Mix
+ template
– template
+ template
– template
Figure 1 Mx3000P multiplex quantitative PCR instrument amplification and dissociation curve plot of a reaction with and
without template DNA. When the amplified products are subjected to dissociation curve analysis, the fluorescence peak
corresponding to the amplicon (centered around 87°C) is distinguishable from the peak due to primer-dimer (centered
around 79°C).
FullVelocity™ SYBR® Green QPCR Master Mix
5
PREPROTOCOL CONSIDERATIONS
cDNA Synthesis and RNA Isolation
High-quality cDNA is essential for successful amplification with the
FullVelocity SYBR Green QPCR master mix. Total and poly(A)+ RNA can
be rapidly isolated and purified using Stratagene's Absolutely RNA®
isolation kits, which are available for nano-, micro- and miniprep scale RNA
purifications (Catalog #400753, #400805, and #400800, respectively). All of
Stratagene’s Absolutely RNA kits include RNase-free DNase and offer a
rapid on-column DNase treatment protocol for removing contaminating
DNA from RNA preparations. cDNA can also be prepared from
Stratagene’s QPCR Human Reference Total RNA (Catalog #750500), a
high-quality control for quantitative PCR gene-expression analysis.
The StrataScript® first strand cDNA synthesis kit (Cat #200420) is
recommended for high efficiency cDNA synthesis from total or poly(A)+
RNA. Up to 10% of the cDNA synthesis reaction (up to 2.5 μl per 25 μl
PCR reaction) can be amplified with FullVelocity SYBR Green master mix.
Primer Design and Concentration
For best results, design primers that amplify PCR products 90–300 bp in
length. The primers should exhibit a melting temperature (Tm) of
approximately 60°C, which is the annealing-extension temperature of the
FullVelocity amplification reaction.
It is critical to minimize the formation of non-specific amplification
products in SYBR Green-based QPCR. This issue becomes more prominent
at low target concentrations. To maximize the sensitivity of the assay, use
the lowest concentration of primers possible without compromising the
efficiency of PCR. It is important to consider both the relative
concentrations of forward and reverse primers and the total primer
concentration. The optimal concentration of the upstream and downstream
PCR primers is the lowest concentration that results in the earliest Ct and an
adequate fluorescence for a given target concentration, with minimal or no
formation of primer-dimer. This concentration should be determined
empirically. Titrating primers over the range of 50–200 nM (each primer)
with a suitable range of template amounts is recommended to identify the
optimal primer concentration for each system. Generally, a primer
concentration of 75–125 nM (each primer) is satisfactory for most systems.
6
FullVelocity™ SYBR® Green QPCR Master Mix
Reference Dye
The passive reference dye included in this kit may be added to compensate
for non-PCR related variations in fluorescence. Fluorescence from the
passive reference dye does not change during the course of the PCR reaction
but provides a stable baseline to which samples are normalized. In this way,
the reference dye compensates for changes in fluorescence between wells
caused by slight volume differences in reaction tubes. The excitation and
emission wavelengths of the reference dye are 584 nm and 612 nm,
respectively. Addition of the reference dye is optional.
Reference Dye Dilution Recommendations
Prepare fresh* dilutions of the reference dye prior to setting up the
reactions, and keep all tubes containing the reference dye protected from
light as much as possible. Make initial dilutions of the reference dye using
nuclease-free PCR-grade H2O. If you are using Stratagene’s Mx4000,
Mx3000P, or Mx3005P instrument, use the reference dye at a 1:500
dilution, resulting in a final concentration of 30 nM. If you are using the
ABI PRISM® 7700 instrument, use the reference dye at a 1:50 dilution,
resulting in a final concentration of 300 nM. For instruments that allow
excitation at ~584 nm (including most tungsten/halogen lamp-based
instruments and instruments equipped with a ~584 nm LED), begin
optimization using the reference dye at a final concentration of 30 nM. For
instruments that do not allow excitation near 584 nm, (including most laserbased instruments) begin optimization using the reference dye at a final
concentration of 300 nM.
Magnesium Chloride Concentration
The optimal magnesium chloride concentration allows for maximum target
amplification with minimal non-specific products and primer-dimers. High
levels of Mg2+ tend to favor the formation of spurious products including
primer-dimers. The 2× FullVelocity SYBR Green QPCR master mix
contains MgCl2 at an optimal concentration of 5 mM, which is suitable for
most targets. Further optimization with additional MgCl2 is normally not
required.
Preparing a Single Reagent Mixture for Multiple Samples
If multiple samples containing the same primers are run, Stratagene
recommends preparing a single mixture of reaction components and then
aliquoting the mixture into individual reaction tubes using a fresh pipet tip
for each addition. Preparing a common mixture facilitates the accurate
dispensing of reagents, minimizes the loss of reagents during pipetting, and
helps to minimize sample-to-sample variation.
Mixing and Pipetting Enzyme Solutions
The FullVelocity SYBR Green master mix should be mixed gently by
inversion without generating bubbles. Bubbles in the final solution may
create optical errors during the sample read.
* The diluted reference dye, if stored in a light-protected tube at 4°C, can be used within the
day for setting up additional assays.
FullVelocity™ SYBR® Green QPCR Master Mix
7
Recommended Control Reactions
Stratagene recommends performing no-template control (NTC) reactions to
identify contamination of reagents or false amplification. NTCs should be
run in at least duplicate reactions.
Preventing Template Cross-Contamination
Take precautions to minimize the potential for carryover of nucleic acids
from one experiment to the next. Use separate work areas and pipettors for
pre- and post-amplification steps. Use positive displacement pipets or
aerosol-resistant pipet tips.
dUTP is included instead of dTTP in the FullVelocity SYBR Green QPCR
master mix. When dUTP replaces dTTP in PCR amplification, treatment
with UNG (Uracil-N-glycosylase, not provided in this kit) can prevent the
subsequent reamplification of dU-containing PCR products. UNG acts on
single- and double-stranded dU-containing DNA by hydrolysis of
uracil-glycosidic bonds at dU-containing DNA sites. When this strategy is
used, carry-over contamination is eliminated while template DNA (DNA
containing T) is left intact.
Fluorescence Detection and Data Acquisition
Fluorescence may be detected either in real-time or at the endpoint of
cycling using a real-time spectrofluorometric thermal cycler. For endpoint
analysis, PCR reactions can be run on any thermal cycler and then analyzed
with any fluorescence plate reader that accommodates PCR tubes and
detects SYBR Green I dye. If using a fluorescence reader, it is
recommended that readings be taken both before and after PCR for
comparison. Stratagene’s real-time PCR instruments can be used for this
purpose. Assemble the FullVelocity SYBR Green reactions and perform a
‘plate-read’ on the instrument before and after thermocycling. The final
ΔCt’s indicate the extent of product formation.
Acquisition of real-time data generated by SYBR Green I binding to dsDNA
should be performed as recommended by the instrument manufacturer. Data
should be collected at the annealing/extension step of the cycling protocol
provided in the Protocols section.
For the Mx3000P or Mx3005P real-time PCR system, use the dissociation
curve included in the default thermal profile for SYBR Green experiments
(Segment 3 of the Original Settings default thermal profile). This
dissociation curve begins with a 1-minute incubation at 95°C to melt the
DNA and then a 30-second incubation at 55°C. This is followed by a ramp
up to 95°C with Allpoints data collection performed during the ramp. Data
collection during the ramp slows the ramp rate to 0.01°C/sec to allow for a
slow melt of the PCR product. Stratagene recommends conducting data
analysis with adaptive baseline and moving average ON, and with
amplification-based threshold OFF. Under these select conditions, each
experiment is analyzed using an adaptive baseline and moving average
selected by the Mx3000P or Mx3005P software.
8
FullVelocity™ SYBR® Green QPCR Master Mix
PROTOCOLS
Preparing the Reactions
Notes
Once the FullVelocity SYBR Green QPCR master mix is thawed,
store the tube on ice while setting up the reactions. Following
initial thawing of the master mix, store the unused portion at 4°C.
Multiple freeze-thaw cycles should be avoided.
It is prudent to set up a no-template control reaction to screen for
contamination of reagents or false amplification.
Consider performing an endogenous control reaction in a
separate tube to distinguish true negative results from PCR
inhibition or failure. For information on the use and production of
endogenous controls for QPCR, see Reference 3.
1.
If the reference dye will be included in the reaction (optional), dilute
the dye solution provided to 1:500 (for Stratagene’s Mx3000P,
Mx3005P, or Mx4000 instruments) or 1:50 (for the ABI PRISM
7700 instrument) using nuclease-free PCR-grade H2O. See the
Reference Dye section of Preprotocol Considerations if using another
type of instrument.
Note
2.
Keep all solutions containing the reference dye protected from
light.
Combine the components listed below in order. Stratagene
recommends preparing a single reagent mixture for replicate
experimental reactions and no-template-controls (plus at least one
reaction volume excess), using multiples of each component listed
below.
Reagent Mixture
Nuclease-free, PCR-grade H2O to adjust the final volume to 25 μl
(including experimental DNA)
X μl of upstream primer (optimized concentration)
X μl of downstream primer (optimized concentration)
0.375 μl of the diluted reference dye (optional)
12.5 μl of 2× FullVelocity SYBR Green QPCR master mix
Note
3.
If the concentration of primer has not yet been optimized
75–125 nM is a suitable starting point.
Gently mix the reagents without creating bubbles (do not vortex) and
then distribute the mixture to the individual PCR reaction tubes.
FullVelocity™ SYBR® Green QPCR Master Mix
9
Add X μl of experimental gDNA, cDNA, or plasmid DNA to each
experimental reaction.
4.
Note
5.
High DNA concentrations may inhibit the PCR reaction.
Stratagene recommends using 10 ng–10 pg genomic DNA,
5
4x10 to 4 copies of plasmid DNA, or cDNA transcribed from
50 ng-5 pg RNA.
Gently mix the reactions without creating bubbles (do not vortex).
Note
6.
Bubbles interfere with fluorescence detection.
Centrifuge the reactions briefly.
PCR Cycling Program
Place the reactions in a QPCR instrument and run the PCR program below.
This protocol has been optimized for 96-well block instruments, such as the
Mx3000P, Mx3005P, and Mx4000 instruments, and delivers excellent
uniformity.
Note
The FullVelocity SYBR Green PCR master mix has been
optimized specifically to support sensitive and reliable
amplification using short denaturation and annealing/extension
times. It is highly recommended that researchers use the provided
cycling conditions to achieve maximum sensitivity and specificity.
Fast Two-Step PCR Program : Plasmid, Genomic DNA, and
cDNA Targets
Cycles
a
10
Duration of cycle
Temperature
1
5 minutes
95°C
40
10 seconds
95°C
30 secondsa
60°C
Set the temperature cycler to detect and report fluorescence during the annealing/
extension step of each cycle.
FullVelocity™ SYBR® Green QPCR Master Mix
Dissociation Program
Mx3000P® and Mx3005P™ Instruments
If using Stratagene’s Mx3000P or Mx3005P instrument, use the default
dissociation curve for SYBR Green experiments. This default profile
dissociation curve begins with a 1-minute incubation at 95°C to melt the
DNA and then a 30-second incubation at 55°C. This is followed by a ramp
up to 95°C with Allpoints data collection performed during the ramp.
Mx4000® Instrument
Incubate the amplified product for 1 minute at 95°C, ramping down to 55°C
at a rate of 0.2°C/sec. For the dissociation curve, complete 81 cycles of
incubation where the temperature is increased by 0.5°C/cycle, beginning at
55°C and ending at 95°C. Set the duration of each cycle to 30 seconds.
Other Instruments
If using another instrument, follow the manufacturer’s guidelines for
generating dissociation curves.
FullVelocity™ SYBR® Green QPCR Master Mix
11
TROUBLESHOOTING
Amplification Plot
Observation
Suggestion
Little or no increase in fluorescence with
cycling
A reagent (primer or template) is missing from the PCR reaction. Set up a
new reaction.
Ensure the correct dilution of reference dye was used.
Optimize the primer concentration.
Ensure that the correct amount of template was used and that the
template sample is of good quality. If unsure, make new serial dilutions of
template before repeating PCR. It may also be possible to check for PCR
inhibitors by adding this target into an assay that is known to work.
Verify that the correct number of cycles was used (40 cycles).
Ensure that the primer Tms are compatible with the annealing/extension
temperature used (60°C).
Analyze the PCR product by gel electrophoresis to determine whether
amplification was successful.
There is increased fluorescence in control
reactions without template
One of the reagents (e.g. primers, master mix, water) has been
contaminated. Follow the procedures outlined in reference 4 to minimize
contamination.
Perform decontamination during amplification by including uracil-Nglycosylase (UNG) in the PCR reaction mix. See Preventing Template
Cross Contamination in Preprotocol Considerations.
The Ct is later than expected when amplifying
from high template amounts
Inhibition may occur at high template amounts (e.g. >100 ng genomic
DNA). Check the DNA template concentration of your sample and verify
that the recommended amount was used. Using a higher initial
denaturation temperature and longer incubation time (98°C for
7 minutes) may minimize inhibition at high genomic DNA template
amounts.
Amplification Plot rises and drops at the
plateau
Some reagent-limiting factor was introduced. Titrate the primer and
template to determine whether the amount of either component needs to
be increased.
Dissociation Plot
Observation
Suggestion
Abundance of primer-dimer and non-specific
PCR products
Check primer design. Verify that the primers do not hybridize to other
target sequences and do not exhibit complementarity at the 3´ ends.
Verify that primers exhibit a Tm of approximately 60°C. Redesign primers.
Perform a primer titration experiment and determine the concentration
that provides the earliest Cts and adequate fluorescence, with minimal
primer-dimer formation.
Verify that the recommended cycling conditions (10 second denaturation,
30 second annealing/extension) are employed.
12
FullVelocity™ SYBR® Green QPCR Master Mix
REFERENCES
1.
2.
Molecular Probes, Inc., at http://www.probes.com/media/pis/mp07567.pdf.
Higuchi, R., Fockler, C., Dollinger, G. and Watson, R. (1993) Biotechnology (N Y)
11(9):1026-30.
Bustin, S. A. (2000) Journal of Molecular Endocrinology 25:169-193.
Kwok, S. and Higuchi, R. (1989) Nature 339(6221):237-8.
3.
4.
ENDNOTES
Absolutely RNA®, Mx3000P®, Mx4000®, and StrataScript® are registered trademarks of
Stratagene in the United States.
FullVelocity, and Mx3005P are trademarks of Stratagene.
ABI PRISM® is a registered trademark of The Perkin-Elmer Corporation.
SYBR® is a registered trademark of Molecular Probes, Inc.
MSDS INFORMATION
The Material Safety Data Sheet (MSDS) information for Stratagene products is provided on Stratagene’s
website at http://www.stratagene.com/MSDS/. Simply enter the catalog number to retrieve any associated
MSDS’s in a print-ready format. MSDS documents are not included with product shipments.
FullVelocity™ SYBR® Green QPCR Master Mix
13
14
FULLVELOCITY™ SYBR® GREEN QPCR MASTER MIX
Catalog #600581, 929581
QUICK-REFERENCE PROTOCOL
•
If the passive reference dye will be included in the reaction (optional), dilute 1:500
(Mx4000®, Mx3000P®, or Mx3005P™ instruments) or 1:50 (ABI PRISM 7700 instrument).
Keep all solutions containing the reference dye protected from light.
•
Thaw the 2× FullVelocity SYBR Green QPCR master mix and store on ice. Keep the master
mix protected from light since it contains SYBR Green I dye. Following initial thawing of the
master mix, store the unused portion at 4°C.
Note
•
Prepare the experimental reactions by adding the following components in order. Prepare a
single reagent mixture for multiple reactions using multiples of each component listed
below.
Nuclease-free, PCR-grade H2O to adjust the final volume to 25 μl
X μl of upstream primer (optimized concentration)
X μl of downstream primer (optimized concentration)
0.375 μl of diluted reference dye (optional)
12.5 μl of 2× FullVelocity SYBR Green QPCR master mix
•
Gently mix the components without creating bubbles (do not vortex), then distribute the
mixture to the individual PCR reaction tubes.
•
Add X μl of experimental gDNA, cDNA, or plasmid DNA to each experimental reaction.
Note
15
Avoid multiple freeze-thaw cycles.
Stratagene recommends using 10 ng–10 pg genomic DNA, 4x105 to 4 copies
plasmid DNA, or cDNA transcribed from 50ng–5pg RNA.
•
Gently mix the reactions without creating bubbles (do not vortex).
•
Centrifuge the reactions briefly.
•
Place the reactions in the instrument and run the PCR program below.
Note
This program is appropriate for most 96-well block instruments, including the Mx3000P,
Mx3005P, and Mx4000 instruments.
Fast PCR Program
Cycles
a
•
Duration of cycle
Temperature
1
5 minutes
95°C
40
10seconds
95°C
30 secondsa
60°C
Set the temperature cycler to detect and report fluorescence during the annealing/
extension step of each cycle.
Run a dissociation curve appropriate to the QPCR instrument.
Stratagene’s Mx3000P and Mx3005P instruments: use the default dissociation curve
for SYBR Green experiments. See the Protocols section for more information.
Stratagene’s Mx4000 instrument: use the following dissociation program. Incubate the
amplified product for 1 minute at 95°C, ramping down to 55°C at a rate of 0.2°C/sec.
Complete 81 cycles of incubation where the temperature is increased by 0.5°C/cycle,
beginning at 55°C and ending at 95°C, with a duration of 30 seconds per cycle.
Other instruments: follow the manufacturer’s guidelines for generating dissociation
curves.
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