qPCR Probe 2X mastermix
CAT# PRODUCT SIZE STORAGE Һڰ ֹ
18100qPCR probe 2X mastermix Kit2X1.25 ml-20C-α
18101qPCR probe 2X mastermix Kit4X1.25 ml-20C-α
18102qPCR probe 2X mastermix Kit10X1.25ml-20C-α
18103qPCR probe 2X mastermix Kit20X1.25ml-20C-α

Description

qPCR 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, Scorpion, Assay On Demand, 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

Reagent
100x50ul
200x50ul
500x50ul
1000x50ul
qPCR probe 2X mastermix Kit
2x1.25ml
4x1.25ml
10x1.25ml
20x1.25ml
25uM ROX dye
100ul
200ul
500ul
2x500ul
 

Kit compatibility

Manufacturer
Model
ROX volume
50ul rxn
Final ROX
concentration
ABI
7000, 7300,
7700, 7900,,
7900HT and
StepOneTM
1.0ul
500nM
7500 and
StepOneTMPlus
0.1ul
50nM
stratagene
Mx4000TM
Mx3000PTM
Mx3005PTM
0.1ul
50nM
 
The qPCR probe 2X mastermix Kit has been optimized for use with all probe chemistries, including PrimeTime Assays,  TaqMan, Scorpion, Assay On Demand, 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 Stability

The qPCR probe 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. When stored under optimum conditions, the reagents are stable for a minimum of 6 months from date of purchase.
 

Quality Control

The qPCR probe 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.
 

Safety Precautions

Harmful if swallowed, Irritating to eyes, respiratory system and skin. Please refer to the material safety data sheet for further information.
 
 

General considerations

 
To 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 60C
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.
 

Procedure

 
The 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 50l final reaction mix and can be scaled accordingly.
Reagent
Volume
Final concentration
2x Probe Master mix
25ul
1x
10uM Forward Primer
0.5ul
250nM
10uM Reverse Primer
0.5ul
250nM
10uM Probe
25uM ROX*
(see Table 1.)
0.5ul
-
100nM
-
H2O
Up to 45ul
 
Template
5ul
 
50ul Final volume
 
(*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 95C. The detection channel on the real-time instrument should be set to acquire at the appropriate wavelength(s).
 

Standard cycling

Cycles
Temperature
Time
Notes
1
*95
*10min
Polymerase activation
40
95
10s
 
60
65s
Acquire at end of step
*Non-variable parameter
 

Fast cycling

Cycles
Temperature
Time
Notes
1
*95
*10min
Polymerase activation
 
95
10s
 
40
60
20s
Acquire at end of step
*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 to 65C.
 

Technical Support

If 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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