Download FluoCycle II™ SYBR® Master Mix

FluoCycle II™ SYBR®
Master Mix
2X Master Mix for Real Time PCR with
SYBR® Green
Cat#
Format
Description
ERD002100BIM
100 reactions
FluoCycle II™ SYBR® Master Mix
ERD002250BIM
250 reactions
FluoCycle II™ SYBR® Master Mix
STORAGE AND STABILITY
1 year at -20°C. The Master Mix tube must be protected from light.
KIT CONTENT
FluoCycle™ II SYBR® Master Mix is a qPCR mix that may be used with any appropriately designed set of primers to detect any DNA or cDNA sequence. FluoCycle™ II SYBR® Master Mix is a 2X amplification mix for Real Time PCR protocols. The detection of PCR product is based on the binding of
the fluorescent dye SYBR®-Green to the double-stranded DNA and fluorescence increases with the amplified PCR product. The kit includes enough
reagents to perform either 100 or 250 x 50 μl reactions. FluoCycle™ II Master Mix SYBR® contains a Hot Start Taq DNA polymerase that can be
activated by an initial step of 5 minutes at 95°C.
Reagent
Size
FluoCycle™ II SYBR® Master Mix*
1.25 ml
(Amber tube)
Quantity
Formulation
2 vials (100 reactions x 50 μl)
100mM KCl, 20mM Tris HCl pH
8.3, 0.02% Tween-20, 0.8mM of
each dNTPs (dATP, dCTP, dGTP,
dTTP), 200 units/ml Taq DNA
polymerase, 8 mM MgCl2, SYBR®
Green I and stabilizers.
5 vials (250 reactions x 50 μl)
100mM KCl, 20mM Tris HCl pH
8.3, 0.02% Tween-20, 0.8mM of
each dNTPs (dATP, dCTP, dGTP,
dTTP), 200 units/ml Taq DNA
polymerase, 8 mM MgCl2, SYBR®
Green I and stabilizers.
*store the tube away from light
NOTES
• SYBR® Green chemistry is an alternate method used to perform real-time PCR analysis. SYBR® Green is a dye binding the Minor Groove of double
stranded DNA. When SYBR® Green dye binds to double stranded DNA, the intensity of the fluorescent emissions increases. As more double stranded amplicons are produced, the SYBR® Green dye signal will increase.
• Primers and probes should be designed within a region including a G/C content ranging from 30% to 80%. Regions with G/C content higher
than 80% might be not completely denatured during thermal cycling, causing lower amplification yield. G/C-rich sequences are also prone to nonspecific interactions decreasing the amplification efficiency and producing a non-specific signal in SYBR® Green dye assays. For the same reason,
primers containing sequences of four or more G/C bases should be avoided.
• To avoid non-specific annealing, primers should not contain more than two C/G repetitions at the last five positions of their 3’ end. Maintaining
the amplified fragment shorter than 150bp while satisfying this requirement might be difficult in case of highly G/C content. In general try to design
primers with the lowest content of G/C repetitions at the 3’ end.
• The primers concentration should be optimised through an initial range from 50nM to 300nM. Because the individual efficiency of the forward
and reverse primer may vary, their respective concentrations must vary to compensate. Therefore all permutations of a selected number of primer
concentrations must be tested. For instance, there are four possibilities of how forward and reverse primer concentrations could be combined for
the following individual concentrations: 50nM, 300nM:
Forward/Reverse: 50/50, 50/300, 300/50, 300/300.
APPLICATION PROTOCOL
We recommend to prepare a PCR mix considering total n. + 1 samples. Add all reagents in a sterile tube and transfer the right volume of the
resulting solution in the final reaction plate or tubes. Add the appropriate volume of DNA template. The reagents of PCR mix have to be mixed
under the following ratio (see table 1 below):
Table 1: reaction set up
Reaction Component
Volume
Final conc. – total quantity
FluoCycle™ II SYBR Master Mix
25 μl
1X
Primers Forward
variable
50-300 nM
Primers Reverse
variable
50-300 nM
DNA template
variable
20 ng - 100 ng
Sterile Water
to reach 50 μl
NA
Total reaction volume
50 μl
®
Transfer the tubes/plate inside the Real Time PCR Platform and run the following program of amplification:
SUGGESTED THERMAL PROFILE
Time
Temperature
Cycles
5 min
95°C
1
15 sec
95°C
45
60 sec*
60°C*
Fluorescent Acquisition
*The exact values for time and temperature must be calculated according to the primers sequences and the amplicon length
SET MELTING CURVE
From 65°C to 95°C increasing of 0.2°C each time
Fluorescence ON
NOTICE TO PURCHASER: LIMITED LICENSE
For Research Use Only (RUO). Diagnostic uses under Roche patents require a separate license from Roche.
SYBR® is a registered trademark of Molecular Probes, Inc.
No right under any patent claim (ex: Patents Nos. 5,210,015 and 5,487,972), no right to perform any patented method, and no right to perform commercial servicesof any kind, including
without limitation reporting the results of purchaser’s activitiesfor a fee or other commercial consideration, is conveyed expressly, by implication, or by estoppel.
Some applications this product is used in may require a license which is not provided by the purchase of this product. Users should obtain the license if required.
Further information on purchasing licenses: Director of Licensing, Applied Biosystems, 850 Lincoln CentreDrive, Foster City, California 94404 USA.
MOLECULAR
BIOLOGY
EuroClone S.p.A.
Via Figino 20/22, 20016 Pero (MI) - Italy
Phone: +39.02.38195.1 - Fax +39.02.38101465
e-mail: [email protected]
www.euroclone.it
I-ERD002_ed0/0813/693_FluoCycleII SYBR
The fluorescence signal registered by the instrument detects the presence of the DNA. Binding of SYBR® Green I to double- stranded DNA is nonspecific and additional testing, such as DNA melting curve analysis, is required to confirm the generation of a specific amplicon. The use of melt
curve analysis eliminates the need for agarose gel electrophoresis because the melting temperature (Tm) of the specific amplicon is analogous to
the detection of an electrophoretic band. When using SYBR® Green I for real-time PCR multiplex reactions, discrimination of amplicons could be
possible, provided the Tm values are sufficiently different.