DescriptionqPCR probe
2X mastermix Kit is a high-performance reagent designed for superior sensitivity
and specificity on all real-time instruments. The kit has been formulated for
use with probe-detection technology, including Primetime Assays, TaqMan¢ç, Scorpion¢ç,
Assay On Demand¢ç, allelic discrimination and molecular beacon
probes. qPCR probe 2X mastermix Kit employs a hot-start DNA polymerase, for
high PCR specificity and sensitivity, Since qPCR probe 2X mastermix Kit
possesses no polymerase activity during reaction set-up, the kit greatly
reduces non-specific amplification including primer-dimer formation. After
pre-heationg, qPCR probe 2X mastermix Kit becomes fully activated and in
conjunction with a specially optimized buffer chemistry, generates reliable and
highly reproducible data on all real-time PCR instruments.
For
ease-of-use and added convenience, qPCR probe 2X mastermix Kit is provided as a
2x mastermix containing all the components necessary for real-time PCR,
including dNTPs and stabilizers. In addition a separate tube of ROX is provided
for optional use. (see notice to purchaser No. 4 in Trademark and Licensing
Information).
Kit Components
Kit compatibility
The qPCR
probe 2X mastermix Kit has been optimized for use with all probe chemistries,
including PrimeTime Assays, TaqMan¢ç,
Scorpion¢ç, Assay On Demand¢ç, allelic discrimination and
molecular beacon probes.
The qPCR
probe 2X mastermix Kit can be used on all real-time PCR instruments.
Optional ROX: Reaction-independent ROX
fluorescence can be measured on the real-time instruments listed below to
normalize the reporter-dye signal during PCR. The qPCR probe 2X mastermix Kit
is supplied with a separate tube of ROX (5-carboxy-X-rhodamine, succinimidyl
ester) at 25uM. Use the following table to determine the appropriate volume of
25uM ROX, per 50ul reaction, to use with the particular real-time instruments.
Storage and StabilityThe qPCR
probe 2X mastermix Kit is shipped on Dry/Blue Ice. All kit components should be
stored at -20¡É upon receipt. Excessive freeze/thawing is not recommended. When
stored under optimum conditions, the reagents are stable for a minimum of 6
months from date of purchase.
Quality ControlThe qPCR
probe 2X mastermix Kit and it¡¯s components are extensively tested for activity,
processivity, efficiency, heat activation, sensitivity, absence of nuclease
contamination and absence of nucleic acid contamination.
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¡ÆC
¡¤ 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_Probe_Kit.pdf (164,433kb) ProcedureThe following are instructions for the use of Dual labeled probes in
real-time PCR. Please refer to the relevant protocols when using other probe
types.
Reaction mix
composition: Prepare a PCR mastermix. The volumes given below are based on
a standard 50¥ìl final reaction mix and can be scaled accordingly.
(*see ROX passive
reference selection above)
If using the ABI Pre-developed
Taqman Assay Reagents(Taqman PDARs) for
allelic discrimination use genomic DNA in the range 10-100ng per 50ul final
reaction mix.
Suggested thermal cycling conditions:The following PCR conditions are
suitable for qPCR probe 2X mastermix Kit with a majority of amplicons and
real-time PCR instruments. However, the cycling conditions can be varied to
suit different probe-based reactions or machine-specific protocols. The
critical step of the PCR is the 10 minute initial activation at 95¡ÆC. The
detection channel on the real-time instrument should be set to acquire at the
appropriate wavelength(s).
Standard cycling
*Non-variable parameter
Fast cycling
*Non-variable parameter
It is
important, when using the ABI Taqman PDARs for allelic discrimination, to
increase the extension temperature in the standard cycling profile from 60¡É to 65¡ÆC.
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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