qPCR Green 2X mastermix with Low ROX
CAT# PRODUCT SIZE STORAGE Һڰ ֹ
18501qPCR Green 2X mastermix with Low ROX Kit4X1.25ml-20C-α
18502qPCR Green 2X mastermix with Low ROX Kit10X1.25 ml-20C-α
18503qPCR Green 2X mastermix with Low ROX Kit20X1.25 ml-20C-α

Description

The qPCR Green 2X mastermix with Low 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 mastermixwith Low 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 with Low 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

 
Reagent 100x50ul 200x50ul 500x50ul 1000x50ul
qPCR Green 2X mastermix Kit with Low ROX 2x1.25ml 4x1.25ml 10x1.25ml 20x1.25ml

Kit compatibility

The qPCR Green 2X mastermix with Low  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.
 
Manufacturer Model
ABI 7500, StepOne PlusTM
Stratagene Mx4000TM, Mx3000PTM, Mx3005PTM

Storage and Stability

The qPCR Green 2X mastermix Low ROX 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 Control

The qPCR Green 2X mastermix Low ROX 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 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 PCR master mix.

 
The volumes given below are based on a standard 50l final reaction mix and can be scaled accordingly.
 

Optional ROX:

 
The qPCR SYBR Green 2X mastermix Low ROX 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.
 
 
Reagent Volume Final concentration
2x SYBR Master Mix with Low ROX 25ul 1x
25uM Forward Primer 0.5ul 250nM
25uM Reverse Primer  0.5ul 250nM
H2O Up to 45ul
Template 5ul
50ul Final volume
 

Suggested thermal cycling conditions

 
The PCR conditions described below are suitable for qPCR SYBR Green 2X mastermix Low ROX 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
 
Cycles Temperature Time Notes
1 *95 *10min Polymerase activation
95 15s
40 55-60 15s Temp. depends on the Tm of primers
72 15s Acquire at end of step
 
*Non-variable parameter

Optional analysis

After the reaction has reached completion refer to the instrument instructions for the option of melt-profile analysis.
 

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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