IDT
IDT
DescriptionThe
qPCR Green 2X mastermix Kit is a high-performance reagent designed for
superior sensitivity and specificity on various real-time PCR instruments. The qPCR Green 2X mastermix Kit employs a hot-start DNA polymerase, for high PCR specificity
and sensitivity. qPCR Green 2X mastermix Kit is inactivate and possesses
no polymerase activity during the reaction set-up, preventing non-specific
amplification including primer-dimer formation.
For
ease-of-use and added convenience, qPCR Green 2X mastermix Kit is provided
as a 2x mastermix containing all the components necessary for real-time PCR,
including the Green I dye, dNTPs, stabilizers and ROX for optional use. As
a ready-to-use premix, only primers and template need to be added.
Kit Components
Kit compatibilityThe
qPCR Green 2X mastermix Kit is optimized for use on the real-time
instruments listed in the following compatibility table.
However
the kit is also compatible with several instruments that do not require the use
of ROX, such as the Qiagen (Corbett) Rotor-GeneTM 6000, the Bio-Rad
CFX96 or the Roche LightCycler 480.
Storage and StabilityThe
qPCR Green 2X mastermix Kit is shipped on Dry/Blue Ice. All kit components
should be stored at -20℃ upon receipt. Excessive freeze/thawing is
not recommended. Since Green I is light sensitive, it is important to
avoid prolonged exposure to light. When stored under optimum conditions, the
reagents are stable for a minimum of 6 months from date of purchase.
Quality ControlThe
qPCR Green 2X mastermix Kit and its components are extensively tested for
activity processivity, efficiency, heat activation, sensitivity, absence of
nuclease contamination and absence of nucleic acid contamination prior to
release.
Safety PrecautionsHarmful
if swallowed, Irritating to eyes, respiratory system and skin. Please refer to
the material safety data sheet for further information.
General considerationsTo
help prevent any carry-over DNA contamination we recommend that separate areas
be maintained for PCR set-up, PCR amplification and any post-PCR gel analysis.
It is essential that any amplified PCR product should not be opened in the PCR
set-up area.
Primers: the sequence and
concentration of primer as well as the amplicon length can be critical for
specific amplification, yield and overall efficiency of any real-time PCR. We
strongly recommend taking the following into consideration when designing and
running your PCR reaction:
· Use primer-design software, such as Scitools (http://eu.idtdna.com/scitools/scitools.aspx),
Primer3 or visual OMPTM (http://frodo.wi.mit.edu/primer3/
and DNA Software, Inc; http://dnasoftware.com/ respectively). Primers should
have a melting temperature (Tm) of approximately 60°C
· Optimal amplicon length should be 50-150bp
· A final primer concentration of 250nM is suitable for most PCR
conditions, however to determine the optimal concentration we recommend a
primer titration in the range of 0.1–1uM
· Use equimolar primer concentrations
· When amplifying from cDNA use gene-specific primers. If possible
use intron-spanning primers to avoid amplification from genomic DNA.
Template: It is important
that the DNA template is suitable for use in PCR in terms of purity and
concentration. Also, the template needs
to be devoid of any contaminating PCR inhibitors (e.g.EDTA). The recommended
amount of template for PCR is dependent upon the type of DNA used. The
following should be considered when using genomic DNA and cDNA templates:
· Genomic DNA: use up to 1g of complex (e.g. eukaryotic)
genomic DNA in a single PCR. We recommend using the Mbiotech Genomic DNA Mini
Kit for high yield and purity from both prokaryotic and eukaryotic sources.
· cDNA: the optimal amount of cDNA to use in
a single PCR is dependent upon the copy number of the target gene. We suggest
using 100ng cDNA per reaction, however it may be necessary to vary this amount.
To perform a two-step RT-PCR, we recommend using the Mbiotech cDNA Synthesis
Kit for reverse transcription of the purified RNA. For high yield and purity of
RNA, use the Mbiotech Total RNA Mini Kit.
MgCl2:
The MgCl2 concentration in the 1x reaction mix
is 3mM, which is optimal for SensiTaq in the majority of real-time PCR
conditions. If necessary, we suggest titrating MgCl2 to a maximum of
5mM.
PCR
controls: It is important to detect the presence of
contaminating DNA that may affect the reliability of the data. Always include a
no template control (NTC), replacing the template with PCR-grade water. When performing
a two-step RT-PCR, set-up a no RT control as the NTC for the PCR.
Mbiotech_SYBR_Kit.pdf (182,147kb) ProcedureReaction mix composition:Prepare a PCR master mix.The volumes given below are based on a standard 50μl final reaction mix and can be scaled accordingly.
Optional ROX:The qPCR SYBR Green 2X mastermix Kit is premixed with ROX (5—carbixyrhodamine, succinymidyl ester), so that where necessary, ROX fluorescence can be optionally detected on certain real-time instruments. If your real-time instrument has the capability of using ROX and you wish to use this option, then this option must be selected by the user in the software.
Suggested thermal cycling conditionsThe PCR conditions described below are suitable for qPCR SYBR Green 2X mastermix Kit for the majority of amplicons and real-time PCR instruments. However, the cycling conditions can be varied to suit customer or machine-specific protocols. The critical step of the PCR is the 10 minute initial activation at 95℃. The detection channel on the real-time instrument should be set to (SYBR) Green or FAM
Optional analysisAfter the reaction has reached completion refer to the instrument instructions for the option of melt-profile analysis.
Technical SupportIf the troubleshooting guide does not solve the difficulty you are experiencing, please contact Technical Support with details of reaction setup, cycling conditions and relevant date. |
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