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                    Products > Real Time PCR > HS Probe qPCR Mix (P2201-P2202-P2203-P2204-P2205)

                    HS Probe qPCR Mix (P2201-P2202-P2203-P2204-P2205)

                    HS Probe qPCR Mix

                    For research use only

                    Components  

                    Component

                    P2201

                    P2202

                    2× HS Probe qPCR Mix

                    1 ml

                    1 ml × 5

                    Nuclease-free Water

                    1 ml

                    1 ml × 5

                     

                    Storage

                    This reagent can be stored for 2 months at 4°C. For longer storage, it should be kept at -20°C.

                     

                    Description

                    HS Probe qPCR Mix is designed for high-performance, high-throughput real-time PCR using sequence-specific fluorogenic probes. 2× HS Probe qPCR Mix contains the components (except primers, DNA template and probe) that necessary to perform PCR. The core component of the mix is OptimusTM Hotstart Taq DNA Polymerase, a hot-start polymerase with chemical modification, which brings high specificity by reducing non-specific products as the enzyme activity is temperature-dependent and is inhibited at room temperature. Combined with well-optimized buffer can significantly improve amplification efficiency. This product is perfectly compatible with common quantitative PCR instruments, such as ABI, Roche, Bio-Rad, etc.

                     

                    Applications

                    ? Probe gene expression analysis

                    ? Probe Low-copy gene detection

                    ? Probe microarray validation

                    ? Probe gene knockdown validation

                     

                    Features

                    ? This kit is suitable for fluorescence quantification by probe method

                    ? This kit is compatible with many real-time systems

                    ? Hot-start technology brings high specificity and reproducible amplification

                     

                    Composition of the 2× HS Probe qPCR Mix

                    KCl, MgCl2, dNTPs, OptimusTM Hotstart Taq DNA Polymerase, and other optimized buffer components.

                     

                    Table of Instrument Guide

                    Instrument

                    Conc. of ROX (100×)

                    ABI? PRISM? 7000, 7700, 7900HT, ABI? 7300 qPCR Systems, GeneAmp? 5700, StepOne?, and the StepOnePlus?

                    1-2% ROX

                     

                    ABI? 7500 qPCR Systems, ViiA? 7, QuantStudio? 12K Flex, Agilent Mx3000P? Mx3005P? and Mx4000?

                    0.2% ROX

                     

                    BioRad iCycler MiniOpticon, Opticon 2, Chromo 4, iQ5; Roche LightCycler 480; MJ Research DNA Engine Opticon 2, Chromo 4; Corbett Rotogene 3000, 6000

                    No ROX

                     

                     

                    Protocol

                    This protocol is intended for use with the BioRad iCycler MiniOpticon, Opticon 2, Chromo 4, iQ5; Roche LightCycler 480; MJ Research DNA Engine Opticon 2, Chromo 4; Corbett Rotogene 3000, 6000. Customers need to prepare ROX Reference Dye, if the instrument needs them.

                     

                    1. Preparation of reaction solution

                    Add the following reagents to the proper thermal cycler reaction tube or plate on ice:

                    Component

                    Volume

                    Final concentration

                    2× HS Probe qPCR Mix

                    10 μl

                    Forward Primer (10μM)

                    0.4 μl

                    0.2 μM

                    Reverse Primer (10μM)

                    0.4 μl

                    0.2 μM

                    Probe (10μM)

                    variable

                    0.1-0.5 μM

                    Template DNA

                    variable

                    0.05-5 ng/μl

                    Water, nuclease-free

                    to 20 μl

                    Note:

                    ? Prepare according to the recommended volume of each instrument. 

                    ? The optimal range for primers is 0.1~1.0 μM. In general, the primers with a final concentration of 0.2 μM work well.

                    ? The concentration of the probe used is related to the Real Time PCR amplification instrument, probe species and types of fluorescent label. Please refer to the instructions when using it. Typically, the final concentration is between 0.1 and 0.5 μM.

                    ? Use 1-10ng cDNA or 10-100ng gDNA for each reaction of 20 μl system.

                    ? Users can increase the amount of the the qPCR Mix when using low-copy gene as template.

                     

                    2. Setup the plate

                    Transfer the reaction mixture to PCR tubes/plates. Reaction volumes can be reduced to 10 μl if the instrument supports a low volume system.

                    Cap or seal the reaction tubes/plates then centrifuge briefly to spin down the contents and eliminate any air bubbles.

                     

                    3. Preform qPCR using the following thermal cycling condition

                    Set the thermal cycling conditions using default PCR thermal cycling conditions specified in the following tables according to the instrument cycling parameters of the specific primers. 

                     

                    Standard 3-step PCR mode: 

                    Initial Denaturation

                    95oC

                    3 min

                    Holding Stage

                    Denaturation

                    95oC

                    10 sec

                    Cycling Stage

                    40 Cycles

                    Annealing*

                    60oC

                    15 sec

                    Extension

                    72oC

                    20 sec

                    *The instrument do signal acquisition at this stage. The usual annealing temperature is 55-65 oC. The annealing temperature is generally set to Tm-5 oC of the primer used, generally not less than 55 oC. (melting temperature, Tm). Set the time of annealing according to the instrument guide.

                     

                    2-step PCR mode:

                    Initial Denaturation

                    95°C

                    3 min

                    Holding Stage

                    Denaturation

                    95°C

                    10 sec

                    Cycling Stage

                    40 Cycles

                    Annealing & Extension*

                    60°C

                    30 sec

                    *The instrument do signal acquisition at the extension stage, and some instruments require more than 30 seconds, such as ABI 7300 for at least 31 seconds and ABI 7500 for at least 34 seconds.

                    If the reaction performs not well, it is recommended to adopt a 3-step amplification procedure.

                     

                    4. Analyze the results

                    Data analysis varies depending on the instrument used. Please refer to your instrument user guide for information.


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