Product Applications
The Lightsaber Green 2 qPCR Master is a ready-to-
Description:
The Lightsaber Green 2 qPCR Master is a high
performance and sensitive reagent which contains a
novel HotStart DNA polymerase specifically
designed for real-time quantitative PCR (qPCR),
optimized buffer components, dNTPs and SYBR
Green I fluorescent dye and Rox reference dye (500
nM). The DNA polymerase in Lightsaber Master is
not only specially engineered to enhance the
performance but also formulated with chemical
modifiers that allows the enzyme shows extremely
low activity before PCR reaction started. By using
such specialized DNA polymerase, non-specific
amplification, which usually caused by mispriming
and/or formation of primer dimers during reaction
setup, is significantly reduced and the overall
reaction efficiency is increased.
The Lightsaber Green 2 qPCR Master is a ready-to-
use reagent mix (except template and primer) to
perform in stringent real-time qPCR reaction
conditions optimally. In combination with a real-
time PCR instrument and suitable PCR primers,
Lightsaber Green 2 qPCR Master is suitable to carry
out high sensitive detection, quantification, and
melting curve analysis of defined DNA sequences.
In principle, the Lightsaber Green 2 qPCR Master
can be used for the amplification and detection of
any DNA or cDNA target, including difficult targets
that are GC-rich or GC-poor. Nevertheless, re-
optimization of the PCR program to the reaction
parameters of a particular real-time PCR instrument
is still required. Refer to general recommendations
of your real-time PCR instrument manual and you
could enjoy high performance qPCR reactions with
Lightsaber Green 2 qPCR Master.
use reagent mix that simplifies the preparation of
qPCR reagents, and suitable applied in:
Gene expression analysis
Low copy gene detection
Absolute quantification
Storage and Stability
Upon receipt, store the Lightsaber Green 2 qPCR
Master protected from light at -20 oC. For short-
term storage (up to 3 month), the product may be
stable at +2 to +8 oC.
When stored under the recommended conditions,
the 2 qPCR Master will retain full activity through
the expiration date printed on the label.
Keep the Lightsaber Green 2 qPCR Master
away from light; be careful not to introduce
contamination.
Avoid repeated freezing and thawing.
The complete PCR mix is stable for up to 48
hrs at +15 to +25 oC. Keep the PCR mix away
from light.
Precautions for use
Minimize exposure of the 2 Master to
direct light, since exposure to direct light for
an extended period of time may result in loss
of fluorescent signal intensity.
Prior to use, make sure the reagent is
thoroughly mixed by gently inverting the
tube several times. Before using, always
ensure that the product has been fully
thawed and mixed well.
Important Parameters
The optimal reaction parameters (concentration of
template DNA and primers, incubation
temperatures, times, and cycle number) depend on
the specific template/primer design and must be
determined in each experiment.
Prepare the template Compatible real-time PCR instruments
Use any template DNA (genomic or cDNA, The Lightsaber Green 2 qPCR Master is proven to
plasmid) suitable for PCR in terms of purity, run experiments on the following:
concentration, and absence of inhibitors. ABI
High concentrations of template may Instrument 7000 StepOne
increase background fluorescence and 7300 StepOne Plus
reduce linearity of standard curves. 7700
For optimal quantitative results, use up to 7900 HT
100 ng complex genomic DNA or 20 ng Protocol
cDNA/plasmid DNA per 20 l reaction. Step 1: Preparation of PCR Master Mix
Store the purified RNA templates at -20 oC or
1 For each 20-l reaction, prepare the following reaction mix:
-70 oC in RNase-free water; Store purified Component Volume Final concentration
DNA templates at -20 oC or -70 oC in Tris-HCl, PCR-grade water Up to 20 l
Forward Primer (10 M) 0.4 l 200 nM
pH 8.0. Reverse Primer (10 M) 0.4 l 200 nM
Template DNA Variable 20 ng
Primer
Lightsaber Green 2 10 l 1X
To take advantage of two-step program, use qPCR Master
Total Volume 20 l
appropriate primer design software to
design primers. Melting temperature (Tm) of The final template concentration varies depending on the copy number of
target DNA present in the template solution. To determine the optimum
primers should be 58 to 60 oC. amount of cDNA template in initial experiments, run undiluted, 1:10

Keep the G/C content in the 40 to 60% range. diluted, and 1:100 diluted cDNA template in parallel.
When the cDNA (reverse transcription solution) is used as a template, it
Avoid runs of an identical nucleotide, should not exceed 10% of the final PCR volume.

especially guanine, where runs of four or

2 Cap or seal the reaction tube/plate and centrifuge briefly to spin
more Gs should be avoided. down the contents and eliminate any air bubbles.
The five nucleotides at the 3end should Step 2: Run the qPCR
have no more than two G and/or C bases.
1 Refer to your real-time PCR instrument guidelines, program the instrument
For optimal results, amplicons of 50 to 200 with the following parameters:
basepairs are strongly recommended. Step Cycle Temperature Hold Detection Remarks
Time
Use PCR primers at a final concentration of Initial 1 95 oC 10 min1 Off Enzyme
denaturation activation
0.1 to 0.4 M. The recommended starting Denaturation 40 95 oC 5 - 10 sec2 Off Amplification

concentration is 0.2 M each. Always use Annealing/ 60 oC 30 sec On and real-time

extension analysis
equimolar primer concentrations. Dissociation According to instrument guidelines On Melting curve

Quality Control
Lightsaber Green 2 qPCR Master is functionally
tested for performance in qPCR generating a
standard curve with human genomic DNA as
template, with a broad dynamic range of 7 orders of
magnitude , a reaction efficiency of 90 105% and
a correlation coefficient 0.99.
1. The 10 min, 95 oC step is required to activate the hotstart Taq DNA Polymerase.
2. In most cases, real-time PCR amplification products do not exceed 300 bp. Therefore,
denaturation at 95 oC for about 5 sec. is usually sufficient.
Note: The qPCR standard protocol is recommended. Try this protocol first,
then optimize the reaction conditions if needed.
2 Place your tubes or plate in the instrument and start the reaction.
3 Analyze the results according to the recommended method for each
instrument.
Step 3: Analyze the results
Data analysis is dependent on experimental design. Refer to your

real-time PCR instrument manual for instruction.

Perform melt curve analysis to identify the presence of primer-

dimers and analyze the specificity of PCR products

Troubleshooting
Observation Possible Causes Solutions

Positive signal in no-template control Contamination 1. If the NTC contains a specific product, discard all reagent, clean all

(NTC) pipettes and prepare fresh stocks of primer.

Primer-dimer formation 1. If the NTC contains nonspecific product, optimize the reaction conditions

may be require:

Increase the combined annealing/extension temperature in 3 oC.

Decrease primer concentration.

Resynthesize or redesign primers.

Poor precision or failed PCR reactions Insufficient cDNA template is 1. Use up to 100 ng complex genomic DNA or 20 ng cDNA/plasmid DNA per

present 20 l reaction.

Quality of cDNA template is 1. Quantify the amount of cDNA template.

poor 2. Prepare fresh cDNA, then repeat the experiment.

Presence of PCR inhibitors 1. Test the cDNA template for the presence of PCR inhibitors.

No amplification results from a sample Inhibitors present in the test 1. Try adding BSA to 0.3% in the PCR.

and the positive control does amplify sample 2. Take extra care with the nucleic acid extraction steps to minimize

carryover of PCR inhibitors.

High CT values Primer-dimer formation 1. Prepare the reaction mixes on ice.

2. To ensure optimal results, run the reaction plate as soon as possible after

completing the reaction setup.

Low Rn or Rn values Extension time is too short 1. Please refer to the protocol for setting in your instrument guidelines.

Primer-dimer formation 1. Prepare the reaction mixes on ice.

2. To ensure optimal results, run the reaction plate as soon as possible after

completing the reaction setup.

High variability across the reaction plate Low quality sealing material 1. Use only high quality clear seals that specifically designed for

fluorescence applications.

Evaporation 1. Make sure that the reaction plate is sealed completely.

PCR efficiency <90% Suboptimal PCR conditions 1. Prepare or purchase fresh qPCR reagent.

Poorly designed primers 1. Design new primers.

PCR efficiency is greater than 110% Excessive primer-dimer 1. Try lower primer concentrations.

Pipetting is inaccurate 1. Test pipette calibration

Multiple Tm peaks (Single product on Repeats sequence or localized 1. If the gel analysis shows that all product is specific, continue to use the

gel) AT or GC-rich in PCR product primers

Multiple Tm peaks (Multiple products Multiple targets in the source 1. Design unique primers.

on gel) material

There is a broad peak at a lower Tm Primer-dimer formation that 1. Prepare the reaction mixes on ice.

than the specific product, especially in anneal at their 3-ends, extend, 1. To ensure optimal results, run the reaction plate as soon as possible after

no-template reactions and then amplify. completing the reaction setup.

Melting temperature of specific product Differences in the buffer 1. Differences the salt concentration in qPCR reagent formulation may affect

is different from other kit composition of qPCR reagent. the melting temperature of the product.