Universal SYBR Green qPCR Protocol

Technology Overview
Assay Considerations
Methods of Quantification
Equipment & Supplies
PCR Mix Selection Guide
Protocol
Troubleshooting
Materials
References

Technology Overview: SYBR Green qPCR

With the development of thermal cyclers incorporating fluorescent detection, PCR has a new, innovative application. In routine PCR, the critical result is the final quantity of amplicon generated after the process. Real-time or Quantitative PCR and RT-PCR use the linearity of DNA amplification to determine absolute or relative amounts of a known sequence in a sample. By using a fluorescent reporter in the reaction, it is possible to measure DNA generation.

Figure 1. Technology Overview: SYBR Green qPCR

In quantitative PCR, DNA amplification is monitored at each cycle of PCR. When the DNA is in the log linear phase of amplification, the amount of fluorescence increases above the background. The point at which the fluorescence becomes measurable is called the Quantification Cycle (C_q) or crossing point. By using multiple dilutions of a known amount of standard DNA, a standard curve can be generated of log concentration against C_q. The amount of DNA or cDNA in an unknown sample can then be calculated from its C_q value.

A) The different phases of the reaction:
Baseline: The initial concentration of template is low; therefore, the fluorescence intensity is too low to be detected and only the background signal is evident.
Exponential: After the target yield has reached the detection threshold, shown as the red threshold line, the course of the reaction can be followed through the exponential phase.
Linear: As the concentration of template increases, the available DNA polymerase concentration reduces and the reaction rate decreases.
Plateau: There is insufficient free enzyme to continue amplification and so after this point, the reaction is at the maximum yield, or the plateau phase.

B) Individual reactions are characterized by the cycle at which fluorescence first rises above the threshold, which is referred to as the Quantification Cycle (C_q). If the starting material is abundant, amplification is observed in earlier cycles, and the C_q is lower. If the starting material is scarce, amplification is observed in later cycles, and the C_q is higher. This correlation between fluorescence, C_q, and amount of amplified product enables quantification of the template over a wide dynamic range.

Real time PCR also lends itself to relative studies. A reaction may be performed using primers unique to each region to be amplified and tagged with different fluorescent dyes. Several commercially available quantitative thermal cyclers include multiple detection channels. In this multiplex system, the amount of target DNA/cDNA can be compared to the amount of a housekeeping sequence e.g. GAPDH or β-actin.

SYBR Green qPCR Applications
Mass Screening	XX
Microarray Validation	XX
Multiple target genes/few samples	X
SNP detection	NR
Allelic discrimination	NR
Pathogen detection	X
Multiplexing	NR
Viral load quantification	NR
Gene expression analysis	X
Gene copy determination	NR
End point genotyping	NR
in vitro quantification	NR

NR= not recommended
X= recommended
XX= preferred method

Assay Considerations

DNA Preparation

The single most important step in assuring success with PCR is high-quality DNA preparation. Integrity and purity of DNA template are essential. Quantitative PCR involves multiple rounds of enzymatic reactions and is, therefore, more sensitive to impurities such as proteins, phenol/chloroform, salts, EDTA, and other chemical solvents. Contaminants can also interfere with fluorescence detection. The ratio of absorbance values at 260 nM and 280 nM gives an estimate of DNA purity. Pure DNA has an A260/A280 ratio of 1.8-2.0. Lower ratios indicate the presence of contaminants such as proteins.

Template

Very few copies of target nucleic acid (equivalent to about 100 pg of gDNA or cDNA) are needed to initiate qPCR. To minimize contamination with reaction inhibitors, the starting template amount should be kept to the minimum required to achieve accurate quantification. When the starting material is RNA, primer design and DNase I treatment will reduce signals that may be generated from gDNA contamination.

Primer Design

Whether using a dsDNA-binding dye or a probe-based detection chemistry, designing high-quality primers is one of the most crucial pre-experimental steps in qPCR. Specific primers for PCR should be designed with the aid of primer design software to eliminate the complications introduced with primer-dimers and secondary structures. Lower primer concentrations decrease the accumulation of primer-dimer formation and nonspecific product formation, which is critical in using SYBR Green I dye in quantitative PCR.

dNTPs

Standard PCR/qPCR mastermixes contain dATP, dCTP, dGTP, and dTTP. However, some mixes are available that replace dTTP with dUTP. Products from previous reactions run with dUTP will contain uracil instead of thymine. These are then susceptible to cleavage by Uracil-DNA-Glycosylase (UNG). Therefore, prior incubation of subsequent reactions with UNG prevents carry-over contamination between reactions. To be effective, all reactions in the laboratory must use dUTP.

Magnesium Concentration

Magnesium chloride (MgCl₂) is necessary for reverse transcriptase, Taq DNA polymerase, and Taq DNA 5’ to 3’ exonuclease activity. Optimum Mg²⁺ concentrations for reactions containing DLP are usually between 3 – 6 mM. Lower magnesium chloride concentrations usually result in the formation of fewer nonspecific products. Some ReadyMix solutions are provided at a 2X concentration of 7 mM magnesium chloride (final concentration 3.5 mM). In some cases, a vial of a 25 mM magnesium chloride solution is provided for further optimization of the final magnesium chloride concentration if necessary. A reaction mix that does not contain MgCl₂ may sometimes be required so that a low concentration can be used, e.g. when using Scorpion Probe detection.

Reverse Transcriptase

A reverse transcriptase enzyme that provides high yields of cDNA, while retaining activity at high temperature, is critical to the success of RT-qPCR. Performance at high temperatures helps to ensure that regions of RNA with significant secondary structure are destabilized and accessible for hybridization and subsequent amplification. When performing one-step RT-qPCR, high-temperature performance allows the use of gene-specific primers with high melting temperatures (T_m), which increases reaction specificity. When performing two step protocols, it is important to ensure that the enzyme results in a linear and proportional yield of cDNA from RNA. Minimizing pipetting can decrease variability. Some ReadyMixes contain primers and other reagents needed to perform RT, for example, ReadyScript^® cDNA Synthesis Mix (RDRT).

Taq DNA Polymerase

As with selecting the most appropriate reverse transcriptase for the RT, selection of the appropriate enzyme is vital. A fundamental problem with natural Taq DNA polymerase is that the enzyme has residual activity at low temperature. Non-specific primer binding leads to non-specific product formation as a result of this residual polymerase activity. Antibody-blocked or chemically-blocked Taq DNA polymerases (‘hot-start’) help to rectify this situation by preventing enzyme activity until the high-temperature, denaturation step begins. Refer to the PCR Mix Selection Guide to define the best hot-start polymerase for your application.

Internal Reference Dye by Instrument Type

Some real-time PCR thermal cyclers require a loading dye such as ROX to control for variability in the optical system and to normalize differences in signal intensity. Likewise, some thermal cyclers require fluorescein to create a virtual background when working with SYBR Green I dye assays (which have very low background). These may be supplied in the ReadyMix or as separate components so the appropriate concentration can be used. In some cases, a vial of internal reference dye is included for reaction normalization. Maximum excitation of this dye is 586 nM and maximum emission is 605 nM. Standard instrument settings for ROX reference dye are satisfactory for the measurement of the internal reference dye. This internal reference dye is necessary for ABI Sequence Detection Systems.

Instruments

Reagents compatible with instruments will need to be selected. Platforms use different normalization dyes, so reagents with compatible normalization dyes will need to be selected (refer to Appendix 1).

Many qPCR instruments have been designed to support a specific range of applications, e.g. contrast the ABI 7900 high throughput capability using automatic loading of 384-well plates with the Illumina Eco instrument that supports a single 48-well plate. The most suitable instrument meets the needs of the research. It is desirable to select an instrument with user friendly software that performs the most desirable functions and has flexibility in terms of data output so that it can easily be manipulated in downstream statistical analysis software packages. This reduces the time required to train personnel and therefore to begin generating results. Additional features that are required include a PCR block that is absolutely uniform (an absolute maximum deviation of 1C_q = 2 fold across 96 wells of replication) and an optical system that excites and detects emission as sensitively and as evenly as possible across a wide range of wavelengths. This allows for a wide choice of fluorophores and enables multiplexing. Other features to consider are the operating costs associated with specific consumables, e.g. if a standard microtiter plate is not used for reactions and also the convenience of loading plates/tubes that are non-standard format.

Controls

A positive control is always helpful to make sure all of the kit components are working properly. A no template/negative control is necessary to determine if contamination is present. A signal in the no template control demonstrates the presence of DNA contamination or primer dimer formation.

Buffer

Buffers or reaction mastermixes typically contain dNTPs, a Taq DNA polymerase, MgCl₂, and stabilizers. SYBR Green I, ROX™, fluorescein, and inert loading dyes may also be included, depending on the detection chemistry, instrument, and reaction requirements. The PCR buffer components and stabilizers are typically proprietary to the manufacturer. If purchased separately, maximum flexibility is possible, since each ingredient can be individually optimized in the reaction. However, in contrast, while purchasing the ingredients together as a mastermix reduces flexibility, it increases batch consistency and convenience while reducing the number of pipetting steps, and hence, the chances of error and contamination.

Data Analysis

Follow the recommendations of the real time instrument used to perform quantitative SYBR Green PCR. The following may help new instrument users. Generally the number of cycles is plotted against the fluorescence. Threshold cycles (C_Ts) or crossing points are used to determine the template amount in each sample. Threshold cycle or crossing point is the first cycle that shows a detectable increase in fluorescence due to the formation of PCR products. The cycles before the crossing point are the baseline cycles. The baseline cycles show no detectable increase in fluorescence due to PCR products. The threshold used to determine when the first detectable increase in fluorescence occurs may also be adjusted manually. The threshold should always be done on a logarithmic amplification plot. In a logarithmic amplification plot the threshold should be set in the log-linear range and not the plateau phase.

Melting Curves

Performing a melting curve analysis at the end of the run will help to analyze only the PCR product of interest. Follow the real time instrument manufacturer’s instructions for melting curve analysis. Successive runs with the same primers can be modified to remove the contribution of primer dimer formation to product signal by collecting data in an additional cycling step, the temperature of which must lie between the already determined dimer and product melting temperatures (T_Ms).

Methods of Quantification

Standard Curves

Standard curves are necessary for both absolute and relative quantification. When generating standard curves, different concentrations of DNA (typically five) should be used to generate a standard curve that will bracket the concentration of the unknown. Each concentration should be run in duplicate.

Absolute and Relative Quantification

This SYBR Green PCR kit may be used to quantify target DNA using either absolute or relative quantification. Absolute quantification techniques are used to determine the amount of target DNA in the initial sample, while relative quantification determines the ratio between the amount of target DNA and a reference amplicon. The ideal reference amplicon would have invariant, constitutive expression. In practice, a housekeeping gene is chosen for this function, but there are other reference choices which better adhere to the above requirements.¹

Absolute quantification uses external standards to determine the absolute amount of target nucleic acid of interest. To remove the differences in quantification due to annealing, the primer binding sites of the external standards must be the same as those in the target sequence. The ideal external standard contains sequences that are the same as the target sequence or which vary only slightly from the target sequence. Equivalent amplification efficiencies between the target and external standard are necessary for absolute quantification. Once a suitable construct or amplicon is identified, a standard curve of external standard dilutions is generated and used to determine the concentrations of unknown target samples.

Relative quantification allows calculation of the ratio between the amount of target template and a reference template in a sample. Since this method measures the amount of target relative to a presumably invariant control, relative qPCR is most often used to measure genetic polymorphism differences, for instance, between tissues or between healthy and diseased samples. The advantage of this technique is that using an internal standard can minimize the variations in sample preparation and handling. When using SYBR systems, the target and internal reference quantification must be run in separate reactions.

The accuracy of relative quantification depends on the appropriate choice of a reference template for standards. Variability of the standard will influence the results and so it is most important that standards be appropriate.¹ Some researchers choose not to run a standard curve and report target quantities as a fraction of the reference, a technique termed comparative quantitation. Alternatively, one may assume that the amplification efficiencies of target and reference are negligible and quantify target based solely on the standard curve determined for the reference sequence. Finally, in the most accurate of the relative quantification techniques, the amplification efficiencies of both the reference and target are measured, and a correction factor is determined. This process, termed normalization,¹ requires a sample containing known concentrations of both target and reference and the generation of two standard curves.

Determination of PCR Reaction Efficiencies

The PCR efficiency between a reference sample and a target sample is determined by preparing a dilution series for each target. The CT values of the reference are subtracted from the target and this difference in CT values is plotted against the logarithm of the template amount. If the resulting slope of the straight line is less than ± 0.1 the amplification efficiencies are judged to be similar.

Equipment

• Quantitative PCR instrument
• Microcentrifuge
• Laminar flow hood for PCR set up (optional)

Supplies

• qPCR SYBR Green Mix – Refer to qPCR Selection Guides (Part 1 and Part 2)
• DNA/cDNA template— cDNA reaction diluted 1:10 to detect a medium to highly expressed targets or 1:2 to 1:5 for rare transcripts or 10 ng to 100 ng gDNA
• Forward and reverse primers diluted to working concentration (10µM working stocks are sufficient for most assays)
  • Predesigned gene expression primers are also available for most model organisms (KiCqStart^® SYBR^® Green Primers, KSPQ12012)
• Sterile filter pipette tips
• Sterile 1.5 mL screw-top microcentrifuge tubes (such as CLS430909)
• PCR tubes, select tubes to match desired format:
  • Individual thin-walled 200 µL PCR tubes (Z374873 or P3114)
  • Plates
    • 96 well plates (Z374903)
    • 384 well plates (Z374911)
  • Plate seals
    • ThermalSeal RTS™ Sealing Films (Z734438)
    • ThermalSeal RT2RR™ film (Z722553)
• PCR grade water (W1754)

SYBR Green PCR Mix Selection Guide – Part 1

complete ReadyMixes with Hot Start (Taq, buffer, dNTPs, reference dye, MgCl2)
Compatible Instruments: Bio-Rad CFX384™ Bio-Rad CFX96™ Bio-Rad MiniOpticon™ BioRad MyiQ™ Bio-Rad/MJ Chromo4™ Bio-Rad/MJ Opticon 2 Bio-Rad/MJ Opticon® Cepheid SmartCycler® Eppendorf Mastercycler® ep realplex Eppendorf Mastercycler® ep realplex2 s Illumina Eco qPCR Qiagen/Corbett Rotor-Gene® 3000 Qiagen/Corbett Rotor-Gene® 6000 Qiagen/Corbett Rotor-Gene® Q Roche LightCycler™ 480	Compatible Instruments: Applied Biosystems 7500 Applied Biosystems 7500 Fast Applied Biosystems ViiA 7 Stratagene Mx3000P® Stratagene Mx3005P™ Stratagene Mx4000™	Compatible Instruments: Applied Biosystems 5700 Applied Biosystems 7000 Applied Biosystems 7300 Applied Biosystems 7700 Applied Biosystems 7900 Applied Biosystems 7900 HT Fast Applied Biosystems 7900HT Applied Biosystems StepOnePlus™ Applied Biosystems StepOne™	Compatible Instruments: BioRad iCycler iQ™ BioRad iQ™5 BioRad MyiQ™	Compatible Instruments: Applied Biosystems 5700 Applied Biosystems 7000 Applied Biosystems 7300 Applied Biosystems 7700 Applied Biosystems 7900 Applied Biosystems 7900 HT Fast Applied Biosystems 7900HT Applied Biosystems StepOnePlus™ Applied Biosystems StepOne™	Compatible Instruments: Bio-Rad CFX384™ Bio-Rad CFX96™ Bio-Rad MiniOpticon™ BioRad MyiQ™ Bio-Rad/MJ Chromo4™ Bio-Rad/MJ Opticon 2 Bio-Rad/MJ Opticon® Cepheid SmartCycler® Eppendorf Mastercycler® ep realplex Eppendorf Mastercycler® ep realplex2 s Illumina Eco qPCR Qiagen/Corbett Rotor-Gene® 3000 Qiagen/Corbett Rotor-Gene® 6000 Qiagen/Corbett Rotor-Gene® Q Roche LightCycler™ 480
KiCqStart® SYBR® Green  qPCR ReadyMix™ (KCQS00)	KiCqStart® SYBR® Green qPCR ReadyMix™ Low Rox (KCQS01)	KiCqStart® SYBR® Green qPCR ReadyMix™ with ROX (KCQS02)	KiCqStart® SYBR® Green qPCR ReadyMix™ for iQ (KCQS03)	SYBR® Green JumpStart™ Taq ReadyMix™ for High Throughput qPCR (S9194)	SYBR® Green JumpStart™ Taq ReadyMix™ for Quantitative PCR, Capillary Formulation (S1816)

SYBR Green PCR Mix Selection Guide – Part 2

ReadyMixes with Hot Start and separate components
Reference Dye separate		MgCl2 separate Reference dye separate
Compatible Instruments: Refer to Appendix 1 to look up optimal reference dye concentration for your instrument
SYBR® Green JumpStart™ Taq ReadyMix™ (S4438)	LuminoCt®SYBR®Green qPCR ReadyMix™ (L6544)	SYBR® Green JumpStart™ Taq ReadyMix™ (S5193)

Protocol

Preparation

1. Place all reaction components on ice.
2. Mix and then briefly centrifuge to collect contents at the bottom of the tube.

Standard SYBR Green I Dye Reaction

Note: We have observed that assays run in KiCqStart ReadyMix are optimal when using a higher primer concentration than in conventional PCR. In the protocols below, we use 450 nM final concentration which we have observed to be the optimal concentration for several independent assays.

1. Prepare enough master mix to run all samples in duplicate.
    a. Be sure to include duplicate No template Negative Controls (NTC).
    b. Select appropriate table below based upon qPCR reagent selected.
    c. Calculate amount of reagents to mix. Add 10% volume to allow for pipetting error
    d. Mix well, avoiding bubbles.

Master Mix for KiCqStart Reagents:

Reactions	Target Final Concentration	Volume per single 20 μL reaction (µL)
2X qPCR mix	1X	10 μL
Forward primer (10 µM stock)	0.45 µM	0.9 μL
Reverse primer (10 µM stock)	0.45 µM	0.9 μL
PCR grade water	-	3.2 μL

Master Mix for Other Complete qPCR ReadyMixes:

Reactions	Target Final Concentration	Volume per single 20 μL reaction (µL)
2X qPCR mix	1X	10 μL
Forward primer (10 µM stock)	0.2 µM	0.4 μL
Reverse primer (10 µM stock)	0.2 µM	0.4 μL
PCR grade water	-	4.2 μL

Master Mix for qPCR Reagents with Separate Components:

Reactions	Target Final Concentration	Volume per single 20 μL reaction (µL)
2X qPCR mix	1X	10 μL
Forward primer (10 µM stock)	0.2 µM	0.4 μL
Reverse primer (10 µM stock)	0.2 µM	0.4 μL
25mM MgCl2 (if separate)	3.5mM	3.5 μL1
Reference dye (if separate)		0 to 0.25 μL2
PCR grade water		4.2 μL

¹Optimal MgCl₂ concentration can range from 1mM to 6mM.
²Refer to Appendix 1 to determine optimal reference dye concentration for your instrument.

2. Setup reactions:
    a. For NTC reactions, add 4 μL of water to the reaction tube.
    b. For experimental reactions, add 4 μL of cDNA solution to the reaction tube.
    c. Centrifuge all tubes briefly. Visually confirm that all tubes or wells contain sample at the bottom at the correct volume.
    d. Carefully aliquot 16 μL of template master mix into each qPCR tube or plate well.
    e. Mix reactions well and spin if needed.
    f. Cap tubes or seal the PCR plate and label (according to instrument requirements). (Make sure the labelling does not obscure instrument excitation/detection light path.)

3. Run samples as per instrument manufacturer recommendations. Examples of standard and fast cycling have been included below.

Standard Cycling Parameters:

	Temp	Time
Initial denaturation	94 °C	2 min
40 cycles:
Denaturation	94 °C	15 sec
Annealing, extension, and read fluorescence	60 °C or 5 °C below lowest primer TM	1 min
(Optional) Hold	4 °C only if products will be run out on a gel

Fast Cycling Parameters:

	Temp (ºC)	Time (s)
Initial denaturation	95	30
40 cycles:
Step 1	95	5
Step 2	58	15
Step 3	72	10

Note: Use standard dissociation curve protocol (data collection).

4. Refer to instrument manual for guidance on how to analyze data.

Appendix 1

Reference dye concentrations recommended for use with instruments. For qPCR reagents with a separate reference dye

Instrument	Final Reference Dye Concentration	µL of Reference Dye (per 20µL reaction)
Applied Biosystems 5700	1X	0.2µL
Applied Biosystems 7000	1X	0.2µL
Applied Biosystems 7300	1X	0.2µL
Applied Biosystems 7500	0.1X	0.02µL
Applied Biosystems 7500 Fast	0.1X	0.02µL
Applied Biosystems 7700	1X	0.2µL
Applied Biosystems 7900	1X	0.2µL
Applied Biosystems 7900 HT Fast	1X	0.2µL
Applied Biosystems 7900HT	1X	0.2µL
Applied Biosystems StepOnePlus™	1X	0.2µL
Applied Biosystems StepOne™	1X	0.2µL
Applied Biosystems ViiA 7	0.1X	0.2µL
Bibby Scientific Techne® Prime Pro 48	not used	-
Bibby Scientific PCRmax® Eco 48	not used	-
Bio-Rad CFX384™	not used	-
Bio-Rad CFX96™	not used	-
Bio-Rad MiniOpticon™	not used	-
Bio-Rad/MJ Chromo4™	not used	-
Bio-Rad/MJ Opticon 2	not used	-
Bio-Rad/MJ Opticon®	not used	-
Cepheid SmartCycler®	not used	-
Eppendorf Mastercycler® ep realplex	not used	-
Eppendorf Mastercycler® ep realplex2 s	not used	-
Qiagen/Corbett Rotor-Gene® 3000	not used	-
Qiagen/Corbett Rotor-Gene® 6000	not used	-
Qiagen/Corbett Rotor-Gene® Q	not used	-
Roche LightCycler™ 480	not used	-
Stratagene Mx3000P®	0.1X	0.02µL
Stratagene Mx3005P™	0.1X	0.02µL
Stratagene Mx4000™	0.1X	0.02µL

Troubleshooting Guide

Problem	Possible Cause	Solution
No PCR product is observed.	A PCR component is missing or degraded.	A positive control should always be run to insure components are functioning. A checklist is also recommended when assembling reactions.
	SYBR is degraded.	Run an agarose gel to analyze the reaction product. If an appropriately sized single band is evident, then detection is faulty. SBYR Green I is light sensitive and must be protected.
	The annealing temperature is too high.	Decrease the annealing temperature in 2-4 °C increments.
	The template is of poor quality.	Evaluate the template integrity by agarose gel electrophoresis. It may be necessary to repurify template using methods that minimize shearing and nicking. There may be no template due to extraction or purification failure.
	Primers are not designed optimally.	Check primer set by running a dilution series on a known template. Reorder or redesign as needed.
	The initial denaturation temperature is too long.	Remove the activation step. JumpStart Taq may be degraded with long (>3 min) initial denaturation times.
	Target template is complex.	In most cases, inherently complex targets are due to unusually high GC content and/or secondary structure. Betaine has been reported to help amplification of high GC content templates at a concentration of 0.8-1.3 M.2
	Reference dye is mismatched	For Rn (normalized fluorescence) plots turn off reference dye. Alternatively, view the raw fluorescence of qPCR amplification plot. Removing normalization often restores plots to the expected shape, allowing the calculation of more reasonable Ct values. Alternatively, one may wish to titrate the reference dye in the reaction. See suggestions in final troubleshooting section.
	Too few cycles are performed.	Increase the number of cycles.
	There is not enough template.	After increasing the number of cycles has shown no success, repeat the reaction with a 10-fold higher concentration of the template.
	PCR product is too long.	The best results are obtained when PCR products are between 100-150 bp and do not exceed 800 bp.
	Mg2+ concentration is suboptimal.	Optimal magnesium chloride concentration can vary based upon assay and can range from 1.5 – 5mM. Use 25 mM MgCl2 (M8787) to perform a magnesium chloride titration within this range.
	Detection was not activated or activated at the wrong step.	Confirm that the acquisition mode is turned on for proper detection. The acquisition mode for SYBR Green detection is “single” and is collected at the extension step or optional detection step.
	Primers are degraded.	Check for primer degradation on a polyacrylamide gel.
	Wrong dye layer chosen.	Ensure the reporter being used is activated in the setup view of the Sequence Detection Software.
	Incorrect values on Y-axis	Change the values on the y-axis. By doubling clicking on ΔRn, the values of the y-axis can be changed.
PCR efficiency is too low (<80%)	The annealing temperature is too low.	Increase the annealing temperature in increments of 2-3 °C.
	Template contains inhibitors	Run a standard curve (log [DNA] vs Cq). If the curve is non-linear at high DNA/cDNA concentrations either revise the DNA/cDNA purification or limit template concentrations to linear range.
	The primers are not designed optimally.	Run a melt curve or agarose gel to check for the presence of multiple amplicons.
	The template is of poor quality.	Evaluate the template integrity by agarose gel electrophoresis. It may be necessary to repurify template using methods that minimize shearing and nicking.
	The initial denaturation temperature is too long.	Remove the activation step. Antibody Hot-Start Taq may be degraded with long (>3 min) initial denaturation times.
	Multiple loci hybridize to the primer set.	Run a melt curve or agarose gel to check for the presence of multiple amplicons. Alternatively, for a sequenced target genome use the NCBI program e-PCR looking for multiple amplicons.
	Pipetting errors cause the fluctuation	Prepare large volume of complete mix and aliquot this into separate reactions. If the variance persists, see below.
	Incorrect reference dye or concentration used for instrument	Refer to Appendix 1 for reference dye concentrations recommended for use with instruments.
	Reference dye is mismatched	For Rn (normalized fluorescence) plots turn off reference dye. Alternatively, view the raw fluorescence of qPCR amplification plot. Removing normalization often restores plots to the expected shape, allowing the calculation of more reasonable Ct values. If normalization is desired, the optimal amount of reference dye must be determined by titration.
Signal is independent of template dilution (multiple products or smeared products)	The annealing temperature is too low.	Increase the annealing temperature in increments of 2-3° C.
	The primers are not designed optimally.	Confirm the accuracy of the sequence information. If the primers are less than 27 nucleotides long, try to lengthen the primers to 27-33 nucleotides. If the primer has a GC content of less than 45%, try to redesign the primers with a GC content of 45-60%.
	The template concentration is too high.	Reduce the concentration of the template in the PCR reaction.
	The primer concentration is too high.	Reduce the primer concentrations in a series of two-fold dilutions (i.e. 0.1 µM, 0.05 µM, 0.025 µM and 0.0125 µM) and subject these trial reactions to PCR.
PCR efficiency is too high.	Multiple loci hybridize to the primer set.	Run a melt curve or agarose gel to check for the presence of multiple amplicons. Alternatively, for a sequenced target genome use the NCBI program e-PCR looking for multiple amplicons.
Technical replicates return widely varied Ct values or data gives uninter-pretable amplifi-cation curves	Pipetting errors cause the fluctuation	Prepare large volume of complete mix and aliquot this into separate reactions. If the variance persists, see below.
	Incorrect reference dye or concentration used for instrument	Refer to Appendix 1 for reference dye concentrations recommended for use with instruments.
Large variability within samples and/or duplicates.	Reactions not well mixed.	Gently vortex and centrifuge reactions.
	Wells not tightly capped or covered.	Tightly cap or cover all wells, even the empty ones. Loose caps can compromise the seal of adjacent wells.
	Initial denaturation is too long.	Decrease initial denaturation to not exceed two minutes.
Multiple PCR products	Primers are not optimally designed.	Confirm the accuracy of the sequence information and the specificity of primer sequence to non-target sequence. Increase the length of the primers to make them more target specific.
	Primers are degraded.	Check for primer degradation on a polyacrylamide gel.
	Mg2+ concentration is suboptimal.	Optimal magnesium chloride concentration can vary based upon assay and can range from 1.5 – 5mM. Use 25 mM MgCl2 (M8787) to perform a magnesium chloride titration within this range.
	The annealing temperature is too low.	Increase the annealing temperature in increments of 2-3 °C.
	Contaminating DNA	Check all reagents for possible contamination and set up reactions in a laminar flow hood to prevent contamination from other reactions.
	Primer-dimers were amplified	Include the optional detection step in the cycling program to avoid detection of primer-dimers.
	The template concentration is too high.	Reduce the concentration of the template in the PCR reaction.
	The primer concentration is too high.	Reduce the primer concentrations in a series of two-fold dilutions (i.e. 0.1 µM, 0.05 µM, 0.025 µM and 0.0125 µM) and subject these trial reactions to PCR.
Linearity of crossing point values do not correspond to log of the template amount	Template amount too high.	Do not exceed the maximum recommended amounts of template DNA.
	Template amount too low.	Increase the amount of DNA.
	Contaminating DNA	Check all reagents for contamination and set up reactions in a laminar flow hood to prevent cross contamination.
	Primer-dimers were amplified	Include the optional detection step in the cycling program to avoid detection of primer-dimers.
Fluorescence is not detected or is variable	SYBR Green ReadyMix was not well mixed.	Mix ReadyMix thoroughly before using to ensure that the SYBR Green will be added in the proper concentration to all capillaries.
	Quantitative PCR instrument is contaminated.	Decontaminate the instrument per the manufacture’s instructions.
	Amplification curves reach a maximum and then decrease at high template amounts	Reduce the number of cycles used for baseline calculation. Baseline correction is overcompensating, and negating signal.
	Improper exposure time	Change the exposure time appropriately if using caps (25) or optical adhesive covers (10).

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