Storage and stability:

Associated products
The SensiFAST SYBR® No-ROX Kit is shipped on dry/blue ice. All kit components should be SensiFAST™ SYBR® No-ROX Kit
stored at -20°C upon receipt. Excessive freeze/thawing is not recommended.
Expiry: Shipping: On dry/blue ice Catalog numbers
Product Description Pack Size Cat No. When stored under the recommended conditions and handled correctly, full activity of the kit is
retained until the expiry date on the outer box label. Batch No.: See vial BIO-98005: 500 x 20 L reactions: 5 x 1 mL

BIO-52065 Quality control: Concentration: See vial BIO-98020: 2000 x 20 L reactions: 4 x 5 mL

10 Preps The SensiFAST SYBR® No-ROX Kit and its components are extensively tested for activity,
Rapid isolation of high-quality genomic DNA from a wide variety of BIO-52066
ISOLATE II Genomic DNA Kit 50 Preps processivity, efficiency, heat activation, sensitivity, absence of nuclease contamination and
samples
250 Preps BIO-52067 absence of nucleic acid contamination.
Safety precautions:
Please refer to the material safety data sheet for further information. Store at –20°C
10 Preps BIO-52068
Rapid isolation of high-quality genomic DNA from a wide variety of BIO-52069 Notes:
ISOLATE II Plant DNA Kit 50 Preps
plant species Research Use Only
250 Preps BIO-52070

10 Preps BIO-52071 Description

Isolation of high-yield and extremely pure total RNA from a variety BIO-52072
ISOLATE II RNA Mini Kit 50 Preps The SensiFAST™ SYBR® No-ROX Kit has been developed for fast, highly reproducible real-time PCR and has been validated on
of samples
250 Preps BIO-52073 commonly used real-time PCR instruments. A combination of the latest advances in buffer chemistry and enhancers, together with an
antibody-mediated hot-start DNA polymerase system, ensures that the SensiFAST SYBR ® No-ROX Kit delivers fast, highly-specific and
ultra-sensitive real-time PCR.
Isolation of high-yield and extremely pure total RNA from a wide 10 Preps BIO-52076
ISOLATE II RNA Plant Kit For ease-of-use and added convenience, SensiFAST SYBR® No-ROX is provided as a 2x mastermix containing all the components
variety of plant species 50 Preps BIO-52077
necessary for real-time PCR, including the SYBR® Green I dye, dNTPs, stabilisers and enhancers. The kit consists of a ready-to-use
premix, only primers and template need to be added.
Quick isolation of high-quality RNA from a variety of sources for 100 mL BIO-38032
TRIsure™
subsequent use in cDNA synthesis 200 mL BIO-38033
Kit components  use primer-design software, such as Primer3 (http://
frodo.wi.mit.edu/primer3/) or visual OMPTM (http://
SensiFAST cDNA Synthesis Fully optimized to generate maximum yields of full-length cDNA 50 Reactions BIO-65053 dnasoftware.com/). Primers should have a melting
500 x 20 µL 2000 x 20 µL
Kit from RNA 250 Reactions BIO-65054 Reagent
reactions reactions temperature (Tm) of approximately 60 °C.

100 g BIO-41026 SensiFAST SYBR® No-ROX mix (2x) 5 x 1ml 4 x 5ml  optimal amplicon length should be 80-200 bp, and should not
Agarose Molecular biology grade agarose exceed 400 bp
500 g BIO-41025
Instrument compatibility  a final primer concentration of 400 nM is suitable for most
The SensiFAST SYBR® No-ROX Kit is compatible with real-time SYBR®-Green based reactions, however to determine the
PCR instruments that do not need a passive reference signal for optimal concentration we recommend titrating in the range
normalization of the data. The SensiFAST SYBR ® No-ROX Kit 0.1-1 μM. The forward and reverse primers concentration
has been optimized for use on the real-time PCR instruments should be equimolar
listed in the following compatibility table.
 when amplifying from cDNA,use of intron spanning primers to
is preferable, to avoid amplification from genomic DNA
Technical support Manufacturer Model Template: it is important that the DNA template is suitable for
If the troubleshooting guide does not solve the difficulty you are use in PCR in terms of purity and concentration. In addition, the
experiencing, please contact Technical Support with details of Opticon™, Opticon2™, MiniOpticon, template must be devoid of any contaminating PCR inhibitors
Bio-Rad
reaction setup, cycling conditions and relevant data. Chromo4™, CFX96, CFX384 (e.g. EDTA). The recommended amount of template for PCR is
dependent upon the type of DNA used. The following points
Email: tech@bioline.com Cepheid SmartCycler™
should be considered when using genomic DNA and cDNA
Qiagen Rotor-Gene™ 3000 & 6000 templates:

Eppendorf Mastercycler® ep realplex  Genomic DNA: use up to 1 g of complex (e.g. eukaryotic)

Trademark and licensing information genomic DNA in a single PCR. We recommend using the
® ®
Roche LightCycler® 480 Bioline ISOLATE II Genomic DNA Kit (BIO-52066) for high
1) Trademarks: SensiFAST™ (Bioline Reagents Ltd), SYBR (Molecular Probes), iCycler™ MyiQ5™, Opticon™, Chromo4™, Miniopticon™, (Bio-Rad), LightCycler
(Roche), StepOne™ (ABI), SmartCycler™ (CEPheid), RotorGene™ (Corbett), RealPlex™ (Eppendorf), Quantica™ (Techne), MX4000 (Stratagene), Thermal Cycler Dice® yield and purity from both prokaryotic and eukaryotic sources.
(Takara)
Techne Quantica®

2) Purchase of this product conveys a licence from Life Technologies to use this SYBR® containing reagent in an end-user RUO assay. Parties wishing to incorporate this BMS Mic  cDNA: the optimal amount of cDNA to use in a single PCR is
SYBR® containing reagent into a downstream kit, should contact Life Technologies for SYBR® Licencing information. dependent upon the copy number of the target gene. We
Takara Thermal Cycler Dice® (TP800) suggest using 100 ng cDNA per reaction, however it may be
necessary to vary this amount. To perform a two-step
General considerations RT-PCR, we recommend using the SensiFAST cDNA
Synthesis Kit (BIO-65053) for reverse transcription of the
To help prevent any carry-over DNA contamination, we purified RNA. For high yield and purity of RNA, use the Bioline
recommend that separate areas are maintained for reaction ISOLATE II RNA Mini Kit (BIO-52072).
set-up, PCR amplification and any post-PCR gel analysis. It is
essential that any tubes containing amplified PCR product are MgCl2: The MgCl2 concentration in the 1x reaction mix is 3 mM.
not opened in the PCR set-up area. In the majority of real-time PCR conditions this is optimal for both
the reverse transcriptase and the hot-start DNA polymerase.
Primers: The specific amplification, yield and overall efficiency
of any real-time PCR can be critically affected by the sequence PCR controls: It is important to detect the presence of
____________________________________________________________________________________________________________________ and concentration of the primers, as well as by the amplicon contaminating DNA that may affect the reliability of the data.
length. We strongly recommend taking the following points into Always include a no-template control (NTC) reaction, replacing
Bioline Reagents Ltd Bioline USA Inc. Bioline GmbH Bioline (Aust) Pty. Ltd Bioline France Meridian Bioscience Asia Pte Ltd
UNITED KINGDOM USA GERMANY AUSTRALIA FRANCE SINGAPORE
consideration when designing and running your real-time PCR: the template with PCR grade water. When performing a two-step
RT-PCR, set up a no-RT control as well as an NTC for the PCR.
Tel: +44 (0)20 8830 5300 Tel: +1 508 880 8990 Tel: +49 (0)337 168 1229 Tel: +61 (0)2 9209 4180 Tel: +33 (0)1 42 56 04 40 Tel: +65 6774 7196
Fax: +44 (0)20 8452 2822 Fax: +1 508 880 8993 Fax: +49 (0)3371 68 1244 Fax: +61 (0)2 9209 4763 Fax: +33 (0)9 70 06 62 10 Fax: +65 6774 6441

Website: www.bioline.com/sensifast email: info@bioline.com PI-50220 V9 Website: www.bioline.com/sensifast email: info@bioline.com

 SensiFAST SYBR® No-ROX Kit is compatible with either three- Troubleshooting guide (Continued)
Procedure
step or two-step cycling:
Reaction mix composition: Prepare a PCR mastermix. The  3-step cycling Problem Possible Cause Recommendation
volumes given below are based on a standard 20 L final
reaction mix and can be scaled accordingly.
Cycles Temp. Time Notes
No
amplification
Final 1 *95 °C *2 min Polymerase activation
Reagent Volume trace
concentration
AND Error in instrument setup Check that the acquisition settings are correct during cycling
2x SensiFAST SYBR® No-ROX Mix 10 L 1x
95 °C 5s Denaturation PCR product
40 60-65 °C 10 s Annealing present on
10 M forward primer 0.8 L 400 nM 72 °C **5-20 s Extension (acquire at end of agarose gel
step)
10 M reverse primer 0.8 L 400 nM
*2 min for cDNA, 3 min for genomic DNA Suboptimal primer design Redesign primers using appropriate software or use validated primers
Template up to 8.4 L ** Not recommended to extend beyond 20 seconds
Test dilution series of primer concentrations until primer dimer/non-specific
Primer concentration too high
H2O As required amplification products disappear
 2-step cycling
20 L Final volume Primer concentration too low Titrate primers in the concentration range of 100 nM - 1 M
Non-specific
Cycles Temp. Time Notes amplification
Sensitivity testing and Ct values: When comparing product Due to the high ionic strength of SensiFAST SYBR® No-ROX Kit it is not recommended
SensiFAST with a mix from another supplier we strongly Primer annealing/extension
to use annealing/extension temperatures below 60 °C. Annealing/extension
1 *95 °C *2 min Polymerase activation AND temperature(s) too low
recommend amplifying from a 10-fold template dilution series. temperature can be increased in steps of 2 °C in the event of non-specific products
Loss of detection at low template concentration is the only direct Primer-dimers
measurement of sensitivity. An early Ct value is not an indication 95 °C 5s Denaturation Template concentration too low Increase template concentration
of good sensitivity, but rather an indication of speed. In some 40
60-65 °C **15-30 s Annealing/extension (acquire at
instances increasing final MgCl2 concentration to 6mM will end of step) Template concentration too high Reduce template concentration until non-specific products disappear
reduce Cts for difficult amplicons.
*2 min for cDNA, 3 min for genomic DNA
Suggested real-time PCR conditions: The following real-time **Not recommended to anneal/extend beyond 30 seconds Extension time too long Reduce extension time to determine whether non-specific products are reduced
PCR conditions are suitable for the SensiFAST SYBR ® No-ROX
Kit with the amplicons of up to 200 bp. However, the cycling Variability Error in reaction set-up Prepare large volume mastermix, vortex thoroughly and aliquot into reaction plate
conditions can be varied to suit different machine-specific Optional analysis: After the reaction has reached completion, between
protocols. It is not recommended to use annealing temperatures refer to the instrument instructions for the option of melt-profile replicates Air bubbles in reaction mix Centrifuge reaction samples/plate prior to running on a real-time PCR instrument
below 60 °C or combined annealing/extension times longer than analysis.
30 seconds. Activation time too short Ensure the reaction is activated for between 1 min and 3 min at 95 °C before cycling

Increasing the extension time may be necessary for amplification products over 200 bp;
Extension time too short
double extension time to determine whether the cycle threshold (C t) is affected
Troubleshooting guide
Annealing temperature too high Decrease annealing temperature in steps of 2 °C

Problem Possible Cause Recommendation Template concentration too low Increase concentration if possible

Late Template with high secondary Increase reverse transcription reaction time up to 30 min
For cDNA templates, make sure SensiFAST SYBR® No-ROX is activated for 2 min at
amplification structure Increase reverse transcription reaction temperature up to 45 °C
Activation time too short 95°C before cycling. For more complex templates such as genomic DNA, increase
trace
inactivation time up to 3 minutes.
Template is degraded Re-isolate template from sample material or use freshly prepared template dilution
Verify that the correct reagent concentrations, volumes, dilutions and storage condi-
Error in protocol setup
tions have been used Suboptimal design of primers Redesign primers using appropriate software or use validated primers

Suboptimal primer design Use primer design software or validated primers. Test primers on a control template Primer concentration too low Increase concentration of primer in 100 nM increments

No amplification Incorrect concentration of MgCl2 concentration is

trace Use primer concentration between 100 nM and 1 M Increase final MgCl2 concentration to 6 mM
primers insufficient
AND Re-isolate your template from the sample material or use freshly prepared template
Template degraded Extension time is too short Increase extension time
No product on dilution
agarose gel PCR
Primers degraded Use newly synthesized primers efficiency Primer concentration too low Increase concentration of primer in 100 nM increments
below 90%
Template contaminated with Further dilute template before PCR or purify template and resuspend it in PCR-grade Suboptimal design of primers Redesign primers using appropriate software or use validated primers
PCR inhibitors water
Template is degraded or Re-isolate template from sample material or use freshly prepared template dilution or
Template concentration too low Increase concentration used PCR contains PCR inhibitors purify template and resuspend it in water
efficiency
above 110% Use melt analysis and 4% agarose gel electrophoresis to confirm presence of
Cycling conditions not optimal Increase extension/annealing times, increase cycle number Non specific amplification and/
non-specific amplification products. See above for preventing/removing non-specific
or primer dimers
products

Website: www.bioline.com/sensifast email: info@bioline.com Website: www.bioline.com/sensifast email: info@bioline.com