qPCR Probe One-step Kit
CAT# PRODUCT SIZE STORAGE Һڰ ֹ
18200One-Step qPCR probe 2X mastermix Kit100 rxn:1 X 1 ml-20C-α
18201One-Step qPCR probe 2X mastermix Kit500 rxn: 5 X 1 ml-20C-α
18202One-Step qPCR probe 2X mastermix Kit, No ROX100 rxn:1 X 1 ml-20C-α
18203One-Step qPCR probe 2X mastermix Kit, No ROX500 rxn: 5 X 1 ml-20C-α
18204One-Step qPCR probe 2X mastermix Kit, Low ROX100 rxn:1 X 1 ml-20C-α
18205One-Step qPCR probe 2X mastermix Kit, Low ROX500 rxn: 5 X 1 ml-20C-α

Description

The Mbiotech Probe One-Step Kit has been formulated for highly reproducible first-strand cDNA synthesis and subsequent real-time PCR in a single tube. A combination of the latest advances in buffer chemistry together with a reverse transcriptase and hot-start DNA polymerase system, ensures that Mbiotech SYBR One-Step Kit produces fast, highly-specific and ultra-sensitive one-step RT-qPCR.
The kit is formulated for use with probe-detection technology, including TaqMan, Scorpions , PrimeTime and molecular beacon probe. A combination of the latest advances in buffer chemistry together with a reverse transcriptase and hot-start DNA polymerase system, ensures that Mbiotech Probe One-Step Kit produces fast, highly-specific and ultra-sensitive one-step RT-qPCR.
 
Mbiotech Probe Hi-ROX One-Step Kit consists of a 2X Hi-ROX One-step mix(ROX for optional use), as well as separate reverse transcriptase and RNase Inhibitor.
Mbiotech Probe Lo-ROX One-Step Kit consists of a 2X Lo-ROX One-step mix(ROX for optional use), as well as separate reverse transcriptase and RNase Inhibitor.
Mbiotech Probe No-ROX One-Step Kit consists of a 2X No-ROX One-step mix, as well as separate reverse transcriptase and RNase Inhibitor.

Kit Components

 
Reagent
100 x 20ul
500 x 20ul
Probe One-Step mix(2x)
1 x 1 ml
5 x 1 ml
RNase Inhibitor
1 x 40 ul
1 x 200 ul
Reverse Transcriptase
1 x 20 ul
1 x 100 ul
DEPC-H2O
1 x 1.8 ml
2 x 1.8 ml

Kit compatibility

The 2x Mbiotech SYBR Hi ROX One-Step Kit has been optimized for use in SYBR Green-based real-time RT-PCR on the real-time instruments listed in the following compatibility table, each of these instruments having the capacity to analyze the real-time PCR data with the passive reference signal either on or off. 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.
Manufacturer
Model
ABI
7000, 7300, 7700, 7900, 7900HT, StepOneTM,  StepOneTM Plus
 
 
 
 
 
The 2x Mbiotech SYBR Lo-ROX One-Step Kit has been optimized for use in SYBR Green-based real-time RT-PCR on the real-time instruments listed in the following compatibility table, each of these instruments having the capacity to analyze the real-time PCR data with the passive reference signal either on or off. 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.
 
Manufacturer
Model
ABI (Invitrogen)
7500, 7500 FAST
Stratagene (Agilent)
Mx4000TM, Mx3000PTM, Mx3005PTM
 
 
 
 
 
 
The 2x Mbiotech SYBR No-ROX One-Step Kit is premixed with SYBR Green I dye and is compatible with real-time instruments that do not need a passive reference signal for normalized for use on the real-time instruments listed in the following compatibility table:
 
Manufacturer
Model
Bio-Rad
OpticonTM, Opticon2TM, MiniOpticon, Chromo4TM, CFX96, CFX384
Cepheid
SmartCyclerTM
Qiagen (Corbett)
Rotor-GeneTM 3000 & 6000
Eppendorf
Realplex
Roche
LightCycler 480
Techne
Quantica

 

 

 

 

 

 

Storage and Stability

The Mbiotech Probe One-Step 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 Mbiotech Probe One-Step 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

Reaction mix composition: Prepare a master mix. The volumes given below are based on a standard 50l final reaction mix and can be scaled accordingly.
 
Reagent
Volume
Final concentration
2x Probe One-Step Mix
10ul
1x
10uM Forward Primer
0.8ul
400nM
10uM Reverse Primer
0.8ul
400nM
10uM Probe
0.2ul
100nM
Reverse Transcriptase
0.2ul
-
RNase Inhibitor (10U/ul)
0.4ul
H2O
up to 16ul
Template 4ul   
     20ul final volume
Suggested RT-PCR conditions: The following RT-PCR conditions are suitable for Mbiotech Probe One-Step 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 RT-PCR is the 10 minute polymerase activation at 95C. The detection channel on the real-time instrument should be set to acquire at the appropriate wavelength(s).

Standard cycling for TaqMan probes

 
Cycles
Temperature
Time
Notes
1
42C
10min
Reverse transcription
1
*95
2min
Polymerase activation
40
95
5s
Denaturation
60
20s Annealing/extension (acquire at end of step)
 
*Non-variable parameter

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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