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Inhibitor Resistant Genotyping PCR ReadyMix™

Product Description

Inhibitor Resistant Genotyping PCR Readymix is a 2X concentrated ready-to-use reaction cocktail for PCR amplification of DNA templates that overcomes inhibitors often present in crude samples extracted from environmental specimens, plant tissues, or animal tissues. It is a versatile and robust PCR reagent that provides highly specific and efficient amplification for genotyping sequence variants including allelic discrimination of single nucleotide polymorphisms (SNPs) using a variety of fluorogenic probe chemistries, including TaqMan® hydrolysis probes. Inhibitor Resistant Genotyping PCR Readymix contains all required reaction components, except primers, probes, and DNA template. The light blue color of the inert tracer dye simplifies reaction assembly in white, or clear, plates and helps to minimize pipetting or mixing errors. It does not interfere with reporter dye fluorescence or affect the stability of the product.

A key component of Inhibitor Resistant Genotyping PCR ReadyMix is an ultra-pure, highly processive thermostable DNA polymerase that is combined with high avidity monoclonal antibodies. This proprietary polymerase mix is highly resistant to PCR inhibitors and provides an extremely stringent automatic hot-start allowing reaction assembly, and temporary storage, at room temperature prior to PCR amplification. Inhibitor Resistant Genotyping PCR Readymix is compatible with both fast and conventional PCR cycling protocols.

Reagents

2X reaction buffer containing optimized concentrations of MgCl2, dNTPs (dATP, dCTP, dGTP, dTTP), hot-start DNA polymerase, ROX reference dye (where applicable), insert blue qPCR dye, and stabilizers.

 

Product Name* Product No. Sku** Reagent Volume
Inhibitor Resistant Genotyping PCR Readymix PIR00 250RXN 2.5mL
1250RXN 12.5mL
5000RXN 50mL
Inhibitor Resistant Genotyping PCR Readymix, low ROX PIR01 250RXN 2.5mL
1250RXN 12.5mL
5000RXN 50mL
Inhibitor Resistant Genotyping PCR Readymix, ROX PIR02 250RXN 2.5mL
1250RXN 12.5mL
5000RXN 50mL

*Refer to Instrument Compatibility Section to select appropriate reagent
**Reaction number based upon a 20µL final reaction volume

Precautions and Disclaimer

This product is for R&D use only, not for drug, household, or other uses. Please consult the Safety Data Sheet for information regarding hazards and safe handling practices.

Storage/Stability

Inhibitor Resistant Genotyping PCR Readymix is stable for 2 years when stored in a constant temperature freezer at –20ºC. For convenience, it may be stored unfrozen at 2 to 8ºC for up to 6 months. After thawing, mix thoroughly before using.

Repeated freezing and thawing does not affect PCR performance.

Instrument Compatibility

Different real-time PCR systems employ different strategies for the normalization of fluorescent signals and correction of well-to-well optical variations. The ROX dye used in these reagents (where noted) has an excitation wavelength of ~490 nm and an emission channel of 605 to 610 nm. It is critical to match the appropriate qPCR reagent to your specific instrument. Please consult the following table, or visit our web site at www.sigmaaldrich.com/pcrselection to find the optimal kit for your instrument platform.

 

Product No. Product Name Compatible Instruments
PIR00 Inhibitor Resistant Genotyping PCR Readymix Bio-Rad CFX384™, Bio-Rad CFX96™, Bio-Rad MiniOpticon™, Bio-Rad/MJ Chromo4™, Bio-Rad/MJ Opticon 2, Bio-Rad/MJ Opticon®, Cepheid SmartCycler®, Eppendorf Mastercycler® ep realplex, Eppendorf Mastercycler® ep realplex2 s, Illumina Eco qPCR, Qiagen/Corbett Rotor-Gene® 3000, Qiagen/Corbett Rotor-Gene® 6000, Qiagen/Corbett Rotor-Gene® Q, Roche LightCycler™ 480
PIR01 Inhibitor Resistant Genotyping PCR Readymix, low ROX Applied Biosystems 5700, Applied Biosystems 7000, Applied Biosystems 7300, Applied Biosystems 7700, Applied Biosystems 7900, Applied Biosystems 7900 HT Fast, Applied Biosystems 7900HT, Applied Biosystems StepOnePlus™, Applied Biosystems StepOne™
PIR02 Inhibitor Resistant Genotyping PCR Readymix, ROX Applied Biosystems 7500, Applied Biosystems 7500 Fast, Applied Biosystems ViiA 7, Stratagene Mx3000P®, Stratagene Mx3005P™, Stratagene Mx4000™

Supplies

Usage Guidelines

  • Primer and Probe Design for Allelic Discrimination PCR
    • The design of highly specific primers and probes is a critical parameter for successful genotyping using fluorogenic probes with PCR. Use of Sigma’s OligoArchitect™ program is encouraged in order to minimize the potential for internal secondary structure and complementation at 3’-ends within each primer, the primer pair, and primer/probe combinations.  (visit sigma.com/probedesignonline)
    • For best results, amplicon size should be between 65 and 200 bp. Optimal results may require titration of primer concentration between 100 and 900 nM.  A final concentration of 300 nM to 400 nM each primer and 100 to 250 nM probe is effective for most applications.
    • Increasing the concentration of the primer that initiates synthesis of the target strand that is complementary to the probe can improve fluorescent signal for some primer/probe systems.
  • Preparation of a reaction cocktail is recommended to reduce pipetting errors and maximize assay precision. Assemble the reaction cocktail with all required components except sample template (genomic DNA or cDNA) and dispense equal aliquots into each reaction tube. Add the DNA template to each reaction as the final step. Addition of samples as 2 to 5-µL volumes will improve assay precision.
  • Sample Template : Inhibitor Resistant Genotyping PCR Readymix can be used with purified genomic DNA or sample lysates. Use 1 to 10 ng of gDNA per reaction well. The optimal amount of lysate will vary for different sample types and lysate procedures. Empirical optimization is often required to determine the optimal dilution range.
  • After sealing each reaction, vortex gently to mix contents. Centrifuge briefly to collect components at the bottom of the reaction tube.

Reaction Assembly

Reagent Volume for 20 μL reaction Final Concentration
Inhibitor Resistant Genotyping PCR Readymix (2X) 10.0 μL 1X
Forward Primer variable 100 – 900 nM (start with 300 nM)
Reverse Primer variable 100 – 900 nM (start with 300 nM)
Probe variable 100 – 250nM (start with 150nM)
Nuclease-free water variable  
Template 2 to 5 μL variable
Final Volume (μL) 20 μL  

For smaller or larger reaction volumes scale all components proportionally.

After sealing each reaction, vortex gently to mix contents. Centrifuge briefly to collect components at the bottom of the reaction tube.

PCR Cycling Protocol

Incubate complete reaction mix in a real-time PCR detection system as follows:

 

  Fast 2-Step Cycling Fast 3-Step Cycling Standard Cycling
Initial denaturation 95°C, 30s* 95°C, 30s* 95ºC, 2-3 min *
PCR cycling (30 - 45 cycles)
95ºC, 3 to 5s 95ºC, 3 to 5s 95ºC, 10 to 15s
  55 to 65ºC, 15s  
60ºC, 20 to 30s † 68 to 72ºC, 10s † 60ºC, 30 to 60s †

The appropriate step for fluorescent data collection varies for different probe assay formats. Data collection for 5’-nuclease probe assays (TaqMan probe) should be carried out at the end of the extension step. Use the annealing step for data collection with hybridization probe assays (HybProbe® FRET hybridization probes, Molecular Beacons, Solaris® qPCR Assays, Scorpions® primers, etc.).

End-point analysis should be carried out at a suitable temperature for your detection probe chemistry.

* Full activation of the DNA polymerase occurs within 10 seconds at 95ºC; however, optimal initial denaturation time is template dependent and will affect qPCR efficiency and sensitivity. Amplification of genomic DNA or supercoiled plasmid DNA targets may require 5 to 10 min at 95ºC to fully denature and fragment the template. Short double-stranded DNA template (PCR product) or single-stranded DNA template, such as cDNA, may require as little as 1s at 95ºC. Use 30s at 95ºC as a general starting point.

† Extension time is dependent upon amplicon length and the minimal data collection time requirement for your qPCR instrument. Use 30s at 60ºC as a general starting point. Some assay designs and/or detection chemistries may require a 3-step cycling protocol for optimal performance. Optimal annealing temperature and time may need to be empirically determined for any given primer set and real-time instrument.

Quality Control

Kit components are free of contaminating DNase and RNase. Inhibitor Resistant Genotyping PCR Readymix is functionally tested for discrimination of a known SNP using a controlled gDNA sample panel, as well as for qPCR of a single copy gene in human gDNA. Kinetic analysis must demonstrate linear resolution over six orders of dynamic range (R2 > 0.990) with a 2-fold discrimination of starting template and a PCR efficiency > 95%.

Materials

     

Limited Label Licenses

The use of certain types of fluorogenic probes with 5' nuclease assays may be covered by one or more of the following US patents and corresponding patent claims outside the US:  U.S. Patent No. 5,538,848, owned by Life Technologies, Corporation. Purchase of this product does not convey rights to practice the methods claimed in U.S. Patent No. 5,538,848, and a license to practice those methods must be obtained from Life Technologies, 850 Lincoln Center Drive, Forest City, California 94404, or by purchase of fluorogenic probes from an authorized source.  No right under any other patent claim and no right to perform commercial services of any kind, including without limitation reporting the results of purchaser's activities for a fee or other commercial consideration, is conveyed expressly, by implication, or by estoppel. This product is for research use only. Diagnostic uses under Roche patents require a separate license from Roche. Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA.

Use of other hybridization probes may be covered by other patents and specific licenses may be required for each technology which is not conveyed by purchase of this product. Consult your probe provider for patent and license information related to each probe chemistry and their use.

Applicable only to products containing Passive Reference The purchase of this product includes a limited, non-transferable right to use the purchased amount of the product to perform Applied Biosystems’ patented Passive Reference Method for the purchaser’s own internal research. No right under any other patent claim and no right to perform commercial services of any kind, including without limitation reporting the results of purchaser’s activities for a fee or other commercial consideration, is conveyed expressly, by implication, or by estoppel. This product is for research use only. For information about these rights or on obtaining additional rights, please contact outlicensing@lifetech.com or Out Licensing, Life Technologies, 5791 Van Allen Way, Carlsbad, California 92008.

ReadyMix is a trademark of Sigma-Aldrich Co. LLC, KiCqStart is a trademark of Quanta BioSciences, TaqMan is a registered trademark of Roche Molecular Systems, Inc.  LightCycler is a registered Trademark of Roche.  Applied Biosystems, StepOne, StepOnePlus, ViiA, and ROX are trademarks Life Technologies Corporation. Stratagene, MX3000P, MX3005P and MX4000 are trademarks of Agilent Technologies, Inc.  Mastercycler is a trademark of Eppendorf.  Rotor-Gene is a registered trademark of Qiagen GmbH.  SmartCycler is a trademark of Cepheid.  CFX96, CFX384, iCycler iQ, iQ5, MyiQ, Opticon, MiniOpticon and Chromo4 are trademarks of Bio-Rad Laboratories.

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