introducing kapa sybr® fast qpcr kits - wur...human b-actin (290 bp) was amplified from 5 log-fold...
TRANSCRIPT
www.kapabiosystems.com
Introducing KAPA™ SYBR® FAST qPCR Kits
KAPA SYBR® FAST qPCR Kits contain a novel DNA polymerase evolved specifically for real-time PCR.
The result is industry-leading performance:
Highest reaction efficiency. Highest signal intensity. Highest sensitivity and linearity. High speed reduces total cycling time.
next generation thinking
in enzyme technology
Containing the first DNA polymerase evolved for real-time PCR.
Amplification Plot
4.000 E+1
9.000 E+1
1.400 E+2
1.900 E+2
2.400 E+2
2.900 E+2
3.400 E+2
∆R
0 5 10 15 20 25 30 35 40Cycle
Kit can be used for this cycler but may require the addition of ROX reference dye
Product Description
KAPA SYBR® FAST qPCR Master Mix is designed for high performance real-time PCR. The kit contains a novel DNA polymerase engineered through a process of molecular evolution. The result is a unique enzyme, specifically evolved for qPCR using SYBR® Green I dye chemistry.
The reaction efficiency of qPCR is critical for accurate and reliable gene quantification. Wild type Taq DNA polymerase is often inhibited by real-time PCR reaction conditions, leading to significant loss in specificity and sensitivity of the assay. The KAPA SYBR® DNA Polymerase has been engineered to perform optimally in stringent qPCR reaction conditions, exhibiting dramatic improvements in signal-to-noise ratio (fluorescence), cycle threshold (CT), linearity, and sensitivity. The novel DNA polymerase and proprietary buffer system enhances the amplification efficiency of difficult templates, including both GC and AT-rich templates.
High speed is achieved through the increased processivity of the engineered KAPA SYBR® DNA Polymerase and the short initial denaturation time required for the activation of the proprietary antibody-mediated hot start.
>> KAPA™SYBR® FAST qPCR Kit Containing the first DNA polymerase evolved for real-time PCR.
Engineered KAPA SYBR® DNA Polymerase and optimized buffer system provides:
Highest reaction efficiency resulting in the earliest cycle thresholds (CT) and extreme sensitivity.
Highest fluorescence resulting in improved signal-to-noise ratio.
Broad dynamic range and linearity.
High speed reduces total cycling time.
Human coagulation factor V (F5) was amplified from log-fold dilutions of human genomic DNA (20 ng - 2 pg) using KAPA SYBR® FAST Master Mix (2x), with standard cycling protocol (green) and fast cycling pro-tocol (blue). Standard cycling conditions: 5 min initial denaturation at 95ºC followed by 40 cycles of 15 sec denaturation at 95ºC and 60 sec combined annealing/extension at 60ºC. Fast cycling conditions: 3 min initial denaturation at 95ºC followed by 40 cycles of 1 sec denaturation at 95ºC and 20 sec combined annealing/extension at 60ºC. Total run time was 1.5 hours for standard cycling and 40 mins for fast cycling.
High speed and high performance
161820222426
101 102 103 104
Ct
Concentration
Standard curveSlope -3.305R2 0.995PCR E�. 101%
1816
2022242628
101100 102 103 104
Ct
Concentration
Standard curveSlope -3.377R2 0.990PCR E�. 98%
Threshold
10-1.0
10-1.2
10-1.4
10-1.6
10-1.8
10-0.8
1.0-0.6
10-0.4
10-0.2
100.0
Norm
. Flu
oro.
5 10 15 20 25 30 35 40Cycle
Amplification Plot
High speed does not compromise performance with the KAPA SYBR® FAST qPCR Kits. As can be seen from the amplification plots and standard curves, high reaction efficiencies are achieved with both fast and standard cycling conditions, even with complex genomic targets. Total run time for the fast cycling protocol was 40 minutes.
Product Applications
KAPA SYBR® FAST qPCR Kits are ideally suited for: Gene expression analysis Microarray validation Low copy gene detection Gene knockdown validation
The KAPA SYBR® FAST qPCR Kit is available as a ready-to-use 2x Master Mix containing all components, except primers and template, for the amplification and detection of DNA in real-time quantitative PCR (qPCR). All KAPA SYBR® qPCR FAST Kits are supplied with antibody-mediated hot start for high specificity and maximum speed.
The KAPA SYBR® FAST qPCR Master Mix(2x) LightCycler™ 480 Kit is formulated for high performance on the Roche LightCycler™ 480 instrument. The KAPA SYBR® FAST qPCR Kit outperforms the competitor using both the recommended cycling protocol and a faster cycling protocol indicating robust, high speed performance.
High performance on Roche LightCycler™ 480
Human B-actin (290 bp) was amplified from 5 log-fold dilutions of human genomic DNA (20 ng - 2 pg) using KAPA SYBR® FAST qPCR Master Mix (2x) LightCycler™ 480 Kit (green) and competitor R (red). Compari-sons were performed according to the competitor R protocol (left panel: 5 min initial denaturation at 95ºC followed by 40 cycles of 10 sec denaturation at 95ºC, 15 sec annealing at 60ºC, 6 sec extension at 72ºC) and KAPA SYBR® FAST protocol (right panel: 3 min initial denaturation at 95ºC followed by 40 cycles of 10 sec denaturation, 15 sec annealing at 60ºC, 1 sec extension at 72ºC).
Superior signal and reaction efficiency
The KAPA SYBR® FAST qPCR Kit outperfoms when compared to other leading fast qPCR kits. The KAPA SYBR® FAST qPCR Kit consistently produces higher signals, earlier Cts, and 100% reaction efficiencies.
Human B-actin (290 bp) was amplified from 5 log-fold dilutions of human genomic DNA (20 ng to 2 pg) using KAPA SYBR® FAST qPCR Master Mix (2x) or fast qPCR competitor kits. KAPA SYBR® FAST qPCR Master Mix (green) vs. Competitor A (orange), competitor Q (purple), or competitor I (blue). Competitor I also amplifies the no template control (NTC). Reactions were performed according to each competitors’ suggested protocol on an ABI Prism® 7900 HT sequence detection system.
Fluor
esce
nce (
483-
533)
Amplification Curves
Cycles21 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40
26.584
24.084
21.584
19.084
16.584
14.084
11.584
9.084
6.584
4.084
1.584
-0.916
KAPA SYBR® FAST Competitor R Slope -3.481 -6.00R2 0.997 0.999PCR E�. 94% 46%
Slope -3.481 -6.00R2 0.997 0.999PCR E�. 94% 46%
Ct
Quantity
4038
34
30
26
22
36
32
28
24
201.50E+00 1.50E+01 1.50E+02 1.50E+03 1.50E+04
Amplification Plot
4.000 E+1
9.000 E+1
1.400 E+2
1.900 E+2
2.400 E+2
2.900 E+2
3.400 E+2
∆R
0 5 10 15 20 25 30 35 40Cycle
Slope -3.331 -3.180R2 0.997 0.994PCR E�. 100% 106%
KAPA SYBR® FAST Competitor A
Ct
Quantity
37
3335
2932
2325
2021
1719
1.50E+00 1.50E+01 1.50E+02 1.50E+03 1.50E+04
Amplification Plot
4.000 E+1
9.000 E+1
1.400 E+2
1.900 E+2
2.400 E+2
2.900 E+2
3.400 E+2
∆R
0 5 10 15 20 25 30 35 40Cycle
Slope -3.331 -3.123R2 0.997 0.983PCR E�. 100% 109%
KAPA SYBR® FAST Competitor Q
Ct
Quantity
37
3335
2932
2325
2021
1719
1.50E+00 1.50E+01 1.50E+02 1.50E+03 1.50E+04
Amplification Plot
5.000 E+1
1.000 E+2
1.500 E+2
2.000 E+2
2.500 E+2
3.000 E+2
3.500 E+2
4.000 E+2
∆R
0 5 10 15 20 25 30 35 40Cycle
NTC
Slope -3.331 -2.881R2 0.997 0.994PCR E�. 100% 122%
KAPA SYBR® FAST Competitor I
Ct
Quantity
37
3335
2932
2325
2021
1719
1.50E+00 1.50E+01 1.50E+02 1.50E+03 1.50E+04
Fluor
esce
nce (
483-
533)
Amplification Curves
Cycles21 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40
24.933
22.433
19.933
17.433
14.933
12.433
9.933
7.433
4.933
2.433
-0.067
KAPA SYBR® FAST Competitor R Slope -3.381 -3.022R2 0.998 0.998PCR E�. 98% 77%
Slope -3.381 -3.022R2 0.998 0.998PCR E�. 98% 77%
Ct
Quantity
4038
34
30
26
22
36
32
28
24
201.50E+00 1.50E+01 1.50E+02 1.50E+03 1.50E+04
www.kapabiosystems.com
Boston, Massachusetts, United States600 West Cummings Park, Suite 5350
Woburn, MA, 01801 U.S.A.Tel: +1 781 497 2933 Fax: +1 781 497 2934
Email: [email protected]
Cape Town, South Africa2nd Floor, Old Warehouse Building, Black River Park,Fir Road, Observatory, 7925 Cape Town, South Africa
Tel: +27 21 448 8200 Fax: +27 21 448 6503Email: [email protected]
Highest fluorescence and earliest CT values
The KAPA SYBR® FAST qPCR Kit exhibits industry-leading performance in both reaction efficiency and signal intensity across a broad range of targets as compared with fast qPCR competitor kits.
Instrument Compatibility
KAPA SYBR® FAST qPCR Kits are supplied as a ready-to-use Master Mix (2x) containing all components, except primers and template, for the amplification and detection of DNA in real-time quantitative PCR (qPCR). The Master Mix (2x) includes hot start KAPA SYBR® DNA polymerase, SYBR® Green I fluorescent dye, MgCl2, dNTPs, reference dye (when applicable), and stabilizers.
For more information on KAPA SYBR® FAST qPCR Kits please visit www.kapabiosystems.comFor technical support please contact: [email protected]
SYBR® is a registered trademark of Molecular Probes, Inc, OregonPRISM®, StepOne™, StepOnePlus™, and GeneAmp® are registered trademarks of Applera Corporation.iCycler®, MyiQ™, iQ™5, Mx3000P®, Mx3005™, Mx4000®, Roto-Gene™, DNA Engine Opticon™, Chromo 4™, LightCycler®, MasterCycler®,and Smart Cycler® are trademarks or registered trademarks of their respective companies.
24
23
22
21
20
19
18
17
16
15
Ct
Comparison of Ct Values
B-actin F5 HPRT1 Amplicon
KAPA SYBR® FAST
Competitor I
Competitor A
Competitor R
Competitor Q
350
300
250
200
150
100
50
0 B-actin F5 HPRT1 Amplicon
Base
line s
ubtra
cted
�uo
resc
ence
Fluorescence Intensity
KAPA SYBR® FAST
Competitor I
Competitor A
Competitor R
Competitor Q
Human B-actin, F5, and HPRT1 targets were amplified from 20 ng (5900 copies) of human genomic DNA on an ABI Prism® 7900 HT sequence detection system. Values represent averages of triplicates. Reactions were performed according to each competitors’ suggested protocol.
KAPA SYBR® FAST qPCR Master Mix (2x) Universal Kit: Includes separate tubes of ROX passive reference dye (high and low)
KAPA SYBR® FAST qPCR Master Mix (2x) ABI Prism® Kit: ROX passive reference dye included in 2x Master Mix
KAPA SYBR® FAST qPCR Master Mix (2x) Bio-Rad iCycler™ Kit: Fluorescein reference dye included in 2x Master Mix
KAPA SYBR® FAST qPCR Master Mix (2x) LightCycler® 480 Kit: Contains no reference dye
KK4600
KK4601
KK4602
KK4603
KK4604
KK4605
KK4606
KK4607
KK4608
KK4609
KK4610
KK4611
1 x 1 ml (100, 20µl reactions)
1 x 5 ml (500, 20µl reactions)
2 x 5 ml (1000, 20µl reactions)
1 x 1 ml (100, 20µl reactions)
1 x 5 ml (500, 20µl reactions)
2 x 5 ml (1000, 20µl reactions)
1 x 1 ml (100, 20µl reactions)
1 x 5 ml (500, 20µl reactions)
2 x 5 ml (1000, 20µl reactions)
1 x 1 ml (100, 20µl reactions)
1 x 5 ml (500, 20µl reactions)
2 x 5 ml (1000, 20µl reactions)
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• •••••••••
•••••••
•••
º º º º º º
• Prefered kit for this cycler º Kit can be used for this cycler but may require the addition of ROX reference dye
•