PACE® Genotyping Master Mix

A universal PCR master mix for allele-specific PCR assays. Precision fluorescent signal generation with consistently high performance at any reaction volume.

About

PACE Genotyping Master Mix uses a novel, universal, fluorescent reporting cassette to produce machine-readable fluorescent signals corresponding to genotypes. PACE compatible genotyping assays are comprised of two competitive allele specific forward primers (which differ in their terminal 3’ bases and unique 5’ tail sequences) and a common, reverse primer.  PACE Genotyping Master Mix is supplied at 2x concentration and with ROX normalising dye at a range of levels to ensure compatibility with your qPCR instrument or reader.

Genotyping assay designs are available from 3CR Bioscience through our free PACE assay-design service; once designed, users can purchase assay primers independently or through 3CR Bioscience using our partial or full-assay validation service.  Pre-existing KASP and Amplifluor assays are fully compatible with the PACE Genotyping Master Mix.

 

REQUIRED COMPONENTS

  • qPCR machine or Thermocycler + Fluorescent plate reader
  • PCR plate or equivalent and appropriate optically clear seal
  • Template DNA
  • PCR-grade water
  • Genotyping assays
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FAQ:

I would like to know more about various versions of PACE® genotyping chemistry

PACE Genotyping Master Mix is our original genotyping master mix and the most cost-effective for high-throughput, cost-driven applications.

PACE 2.0 Genotyping Master Mix is an enhanced version of PACE Genotyping Master Mix. PACE 2.0 Genotyping Master Mix has a higher signal-to-noise ratio and produces tighter groups on genotyping plots. It is also inhibitor-resistant making it suitable for crudely extracted DNA, and provides the customer the ability to monitor the reaction in real-time.

PACE Multiplex Master Mix enables four fluor amplification and detection. It enables customers to run two SNP assays per well or alternatively, one reference gene and a further three genes of interest. To be able to utilise this master mix, you will require a plate reader capable of reading FAM, HEX, ATTO 550, ATTO 647N and ROX (wavelengths of which can be found in our user manual).

PACE OneStep RT-PCR Master Mix is a one-step RT-PCR PACE formulation, enabling the analysis of RNA samples directly, suitable for both real-time and endpoint detection.

Do I need to use purified DNA for PACE genotyping?

Purification of DNA will generally lead to better genotyping data quality than using a crude extract, but PACE Genotyping Master Mix works well with most crude DNA extraction methods. For some crops such as palm oil, where inhibitor concentration is high in crude extracts, we recommend the use of PACE 2.0 Genotyping Master Mix, which contains inhibitor-resistant components.

My DNA samples are crudely extracted, which version of PACE Genotyping Master Mix should I use?

PACE Genotyping Master Mix works well with most crude DNA extraction methods such as Hot Shot for most crop types. Dilution of the crude extracts should minimise the inhibitor concentration whilst still containing enough DNA for PCR to proceed.

However, for crude DNA extraction of some crops / tissues it may not be possible to derive such a dilution window due to the greater presence of PCR inhibitors. Where this is the case, we would recommend use of our inhibitor-resistant master mix, PACE 2.0 Genotyping Master Mix.

Does PACE Genotyping Master Mix work with PACE®, KASP™ and Amplifluor® assays?

Yes. Assays used for PACE, KASP and Amplifluor utilise the same 5’ tail sequences and are directly compatible with PACE and PACE 2.0 Genotyping Master Mix.

Can I use PACE for insertion / deletion (Indel) genotyping?

Yes. PACE works very well for insertion / deletion genotyping.

What concentration of DNA samples should be used with PACE Genotyping Master Mix?

PACE Genotyping Master Mix works well across a large range of DNA concentrations. We recommend a final DNA concentration of 2-5 ng / µL for optimal results, though good results can be obtained outside of that range.

What controls should I use for genotyping?

Negative controls are very important to establish that the amplification being seen is real. Negative controls should always be used and should consist of the buffer the DNA samples are hydrated in.

Positive controls can be included to increase confidence that the correct genotypes are assigned to the samples, though this is not necessary in all cases. Positive controls should be samples of known genotype and are generally of more use when assessing a very low frequency variant.

What are the optimal storage conditions for PACE genotyping master mix?

PACE and PACE 2.0 Genotyping Master Mix can be stored at 4°C for up to two weeks. However, for longer term storage we recommend storing the master mix at -20°C in smaller aliquots to minimise freeze / thaw cycles.

What is the shelf life of PACE Genotyping Master mix?

PACE and PACE 2.0 Genotyping Master Mix is stable for up to two weeks at 4°C and two years when stored at -20°C / -80°C.

Can I use 5’ nuclease probe-based (e.g., TaqMan™) assays with PACE Genotyping Master Mix?

No, PACE Genotyping Master Mix should be used with PACE assays. Similarly, PACE assays cannot be used with ProbeSure Genotyping Master Mix.

Can I dilute 3cr Bioscience master mixes below 1x to make them go further?

We have encountered customers using master mixes at final concentrations below the intended 1x. This may work in some instances. Use of any of our master mixes below (or indeed above) 1x final concentration is not recommended as it could potentially lead to erroneous results.  All our master mixes have been developed such that their components are optimal for their intended use at 1x concentration in the final reaction mix.

Do I need to optimise PACE cycling conditions for different assays?

The very large majority of assays will work well with the standard cycling conditions listed in the user guides. There are some exceptions, such as assays with a very high GC content or where homology is an issue. If you experience any issues with cycling conditions for difficult assays, please contact our Technical Support team. We are very experienced in optimising assay designs for difficult areas.

What software should I use for analysis of PACE genotyping data?

If you are using a qPCR instrument, many of the software packages that are supplied with the instrument can analyse PACE genotyping reaction data in real-time and endpoint modes.

For some qPCR instruments, and plate readers, where the supplied software cannot perform endpoint genotyping analysis directly, it is possible to use MS Excel or alternative software packages for the analysis.

Which ROX concentration do I require in my Genotyping Master Mix?

We manufacture PACE and ProbeSure Genotyping Master Mixes with different ROX levels. PACE Genotyping Master Mix is available as a high ROX (500 nM), low ROX (25 nM), standard ROX (150 nM) and no ROX version. ProbeSure Genotyping Master Mix is available as a high ROX (500 nM), low ROX (25 nM) and no ROX version.

If you are unsure about which ROX level is required for your qPCR machine or plate reader, please refer to the PACE ROX Instrument Compatibility List and ProbeSure ROX Instrument Compatibility Lists, or contact 3CR Bioscience technical support support@3crbio.com and we will advise you.

Do I have to buy PACE assays from 3cr Bioscience?

No, we offer a free assay design service that enables you to order primers and prepare assays yourself. Please see our free assay design service. Alternatively, if you already have KASP™ or Amplifluor® assays, you can use these.

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MEET OUR TEAM

Steve Asquith Managing Director
Steve began his career in the Genetics Division of GlaxoSmithKline, as part of the team establishing GSK’s high-throughput core genotyping laboratory. Steve joined KBioscience when it was first founded in 2002 and was a key driver in taking the company from a small start-up to a multi-national service laboratory, quickly growing the company’s revenue to over $7.5M p.a. Following the acquisition of Kbioscience by LGC in 2011, Steve was appointed Global Director of Operations for LGC Genomics, responsible for over 100 staff in Europe and N. America, successfully elevating the genotyping products and service business. Steve held a crucial leadership role until he left in 2016. In 2017 Steve joined forces with John Holme to create 3CR Bioscience, a new company with a mission to deliver outstanding, customer-focused genotyping products with innovation and affordability at its core.
Dr. John Holme Technical Director
John joined KBioscience shortly after it was founded, in 2003, and became Head of Technical Development, building the company's genotyping and DNA extraction product portfolio and service delivery until 2011 when it was acquired by LGC. Post-acquisition, John was appointed Head of Technical Group for LGC Genomics, in charge of all Research & Development and Technical Support activities for the company. In this role John continued to build on the high-quality products and services provided to the companies growing customer base. During the 19 years John has worked in commercial R&D, he has co-invented numerous highly successful products including PACE®, ProbeSure, KASP™, KlearKall, KlearGene, KlearAmp and KlearTaq™, creating breakthrough offerings in genotyping and extraction and generating huge revenues for the companies he has worked in. In 2017, he joined forces with Steve Asquith and started 3CR Bioscience. John is dedicated to developing outstanding, innovative genotyping products and providing the very best technical support to customers globally.
Dr. Nisha JainOperations Director
Nisha has been innovating since the start of her career at Geneform Technologies developing Iso-thermal Genotyping Technologies. Nisha joined KBioscience in 2008, as Senior R&D Scientist and key account Technical Support Scientist, developing KASP and Klearkall performance and coinventing two further versions of KASP. Nisha has more than 15 years’ experience working in molecular biology and genotyping technologies, with extensive experience in the areas of R&D, Quality Assurance and Customer Technical Support. She has technically assisted many giants of the industry with their protocol development and troubleshooting and continues to deliver high-quality support and guidance. In 2018, Nisha joined 3CR Bioscience as Operations Director where she continues to develop PACE and ProbeSure for an increasing range of applications, and to grow 3CR Bioscience’s new product pipeline. Nisha is dedicated to developing outstanding, innovative genotyping products and providing the very best technical support to customers globally.
Nazma Saffin General Manager
For 20 years Nazma Saffin has worked and gained extensive expertise within the genotyping sector. Working at Kbioscience and then LGC, she has held operational leadership posts responsible for manufacturing and laboratory services. With experience of ISO 9001 implementation, production scale up and LEAN operations, Nazma has successfully led highly profitable production departments. Joining 3CR Bioscience in 2022, Nazma is committed to delivering operational excellence.
Jon Curtis Non-Executive Chair
After 8 years in The Royal Air Force, Jon moved to the Imperial Cancer Research Fund where he pioneered the use of ultra high-throughput genomic automation, capable of 46,000 PCRs per hour. In the 1990’s Jon joined GlaxoSmithKline, implementing a high-throughput genomics platform into their drug discovery pipeline. Whilst there he also developed acoustic mixing into compound management, becoming the gold standard across pharma. Jon developed the world’s first commercially viable 1536-well PCR plates, automated thermal & laser plate-sealer, plus automated liquid-handling & tip washing tools to reduce waste and costs. In 2002 Jon co-founded KBioscience with Phil Robinson, utilising ultra high-throughput PCR instrumentation & a suite of automation tools to create the company’s SNPline robotic platform, with a capacity of 250,000 PCRs/day. The business was underpinned by their ground-breaking patented genotyping chemistry, KASP™, which has over 10,000 scientific papers to date. In November 2022 Jon joined 3CR Bioscience acting as an advisor bringing his commercial and scientific experience to the company.