3CR SUPPORT​

Questions about our products? You can find users guides, quality, and safety documents for all our reagents available on this page. Our FAQ section will guide you through some common customer questions. We’ve tried to cover everything, but it you can’t find what you need, please contact us on support@3crbio.com.

Filter documents by:

Product

Technical Notes

No related technical notes found.

Publications

Frequently Asked Questions

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.

What types of probes can I use with ProbeSure Genotyping Master Mix?

Any dual-labelled probe with a fluorescent label at the 5′ end and a quencher at the 3′ end, such as TaqMan™, ZEN™ probes and BHQ® and BHQplus® probes, can all be used with ProbeSure 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.

Can I use PACE assays with ProbeSure Genotyping Master Mix?

No, ProbeSure Genotyping Master Mix can only be used with 5′ nuclease probe-based (e.g., TaqMan) assays only. Similarly, 5′ nuclease assays cannot be used with PACE Genotyping Master Mix.

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.

My product arrived defrosted, can I still use it?

Both PACE and ProbeSure Genotyping Master Mixes are stable during shipment if kept cold. If you receive the products defrosted but the contents are still cold, the reagents should still be perfectly OK to use. Feel free to contact 3CR Bioscience if there are any quality issues.

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.

Can I use PACE genotyping master mix to generate real-time data?

Yes, PACE 2.0 Genotyping Master Mix is suitable for real-time applications.

Do I need to optimise the PACE cycling conditions for different thermal cyclers?

This should not be necessary. Cycling conditions are described in detail in the PACE® Genotyping Master Mix User Guide.

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.

Can I use wet or dried DNA samples for genotyping?

All our master mixes can be used with hydrated or dry DNA samples. If high numbers of samples are to be genotyped in one run, drying the DNA samples can improve the resulting genotype data.

To dry the DNA, once dispensed into a PCR plate, the plate should be centrifuged to ensure the samples are all in the bottom of the wells, and then placed in a laboratory fan oven for one hour at 55˚C, or until the samples have visibly dried.  DNA dried onto a PCR plate is stable long term at ambient temperature.  

How do I know if my reader is compatible with PACE multiplex master mix?

You will require a plate reader capable of reading FAM, HEX, ATTO 550, ATTO 647N and ROX (the wavelengths of which can be found in our PACE Multiplex Master Mix User Guide). It is important that filters used in the plate reader have narrow bandwidths (10 nm) to avoid cross fluorescence. Alternatively, plate readers using monochromators capable of using at least five channels with 10 nm bandwidth will also work.

Can I use PACE Multiplex Master Mix on my qPCR instrument?

PACE Multiplex Master Mix contains uses the fluorophores FAM, HEX, ATTO 550, ATTO 647N. You can use PACE Multiplex Master Mix on any qPCR instrument or with any plate reader that can detect these fluorophores. Please refer to the PACE Multiplex Master Mix User Guide and your instrument manufacturer first.

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 you supply ROX normalising dye separately?

No. Accurate titration of ROX levels in any master mix is required during the bulk product manufacturing process, at large scale. Adding ROX to individual master mix bottles, for example prior to a genotyping experiment, is prone to very high levels of error compromising the performance of the mix, and is therefore not included as an option.

How do I submit my sequences for the free PACE assay design service?

You can send your PACE assay design request directly to 3CR Bioscience by completing the PACE assay design template and emailing it to support@3crbio.com.

Do I need to submit sequences in a specific format for PACE assay design services?

Our PACE assay design template contains information and guidance on submitting your assay sequences. Please read, then fill in the template and send it to support@3crbio.com.

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.

How long does it take to design and validate a PACE assay?

We have a 24 hour turnaround on assay designs. We also offer a full or partial validation service were we order and validate your assays before shipping them to you. This will take longer, and will depend upon the size and complexity of the project. Our typical turnaround time for validated assays is 2-4 weeks after receiving the DNA samples.

What sequence information do you need to design the primers for a specific SNP, Indel or other variant?

We request 50-100bp of sequence information upstream and downstream of the target SNP. We do not need a genome reference and coordinates but if you have any specific information about the sequence that is relevant to primer design such as homologous regions etc., please provide this as it will help us in designing primers that are specific to the target.

Can I use PACE for presence / absence or adventitious presence assays?

Yes. However, when we design assays for these applications, they are not competitive and so do not use two allele-specific forward primers and one common reverse primer like standard genotyping assay. Instead, we design one assay comprised of a (5’ tailed) forward primer and a reverse primer for the detection of one sequence and another assay of the same type for the detection of the other sequence. The two assays report with different fluorophores (FAM and HEX).

My presence / absence assays have two reverse primers. How do I assemble PACE assays with this configuration?

You should use both forward primers (i.e., those with a 5’ tail sequence) at 12µM and both reverse primers at 30 µM final concentration in the 72x PACE assay mix.

Is it possible to use the PACE genotyping for pathogen detection?

Yes, we offer custom pathogen detection kits for this type of application. Please contact support@3crbio.com with the relevant sequence information.

Do you need genome coordinates to design assay primers?

No, we do not need genome coordinates.

What is the best way to ship DNA samples to 3cr Bioscience for PACE assay validation?

You do not need to use dry ice for shipping DNA samples. You can ship samples using blue ice blocks. However please ensure that the DNA samples are secured to avoid any leakage or contamination in transit. If sending DNA in plates, seal the plate securely using a heat seal and then wrap the sealed plate in bubble wrap to prevent physical damage.

Do I need to send my PACE assay design request to IDT or 3CR? Is there any difference in the assay designs provided?

You can send your PACE assay design request directly to 3CR by sending a PACE design template to support@3crbio.com or to IDT. There is no difference between the assay designs as all PACE design requests sent to IDT are carried out by 3CR Bioscience Technical Support team in any case.

I have received a lyophilized PACE genotyping assay from IDT. How much TE buffer or PCR-grade water do I need to add?

You will need to add 100 µL of TE buffer (10 mM Tris pH 8.0, 0.1 mM EDTA) or PCR-grade water to each tube of lyophilized PACE assay mix to achieve 72x assay mix.

How much flanking sequence is required either side of my SNP of interest for PACE assay design?

We recommend 50-100bp flanking region on each side of the SNP, Indel or other variant of interest. This provides our assay design software flexibility to generate optimal primer set. If you have less that 50bp of flanking sequence then it may still be possible to design primers, but they might not be optimal.

How do I get PACE primers designed for presence / absence assays or adventitious presence assays?

Please contact support@3crbio.com with the relevant sequence information. We will discuss your requirements and make the necessary assay designs.

I cannot provide samples for PACE assay validation. Can I still use PACE assay validation services?

Yes, you can. We will order IDT gBlocksTM templates on which to test your assays. There will be an additional charge for the gBlocks.

Do you also offer a free assay design service for 5’ nuclease probe-based (e.g., TaqMan™) assays for use with ProbeSure genotyping master mix?

No. However, 5’ nuclease assays are available from a range of manufacturers, such as IDT.

Your reagent pack sizes do not suit my needs. Can I have a custom pack size?

We are happy to manufacture a custom pack size for users that are planning to make a regular order of that pack size.

What is a fluorescent reader gain setting and why is it important?

The fluorescent reader gain setting determines the amplification of the incoming fluorescent signal. It is important to use gain settings in the correct ranges. Gain set too low will not capture as much signal as possible, leading to poorer quality data. Gain set too high will saturate the reader leading to meaningless results, which are not always easy to spot and can therefore lead to incorrect data. We advise first running a test with the reader set for optimised gain, and then use the values suggested by the reader.

Can the ProbeSure Genotyping Master Mix be used with intercalating dyes in real-time PCR?

No. ProbeSure Genotyping Master Mix is not designed for use with intercalating dyes. We do not currently make a product specifically or this application, though it is something we’d consider if enough customers request it.

Are 3cr Bioscience 384-well plates barcoded?

No, these plates are not supplied with a barcode. You will need to source your barcode separately, if required.

Can 3cr Bioscience 384-well plates be autoclaved?

No, our 384-well plates will warp under autoclave conditions.

Do you supply 384-well sample plates to test?

Yes, we can send a sample plate for you to test. Please email your contact details to orders@3crbio.com, along with the part code of the plate.

How does PACE OneStep RT-PCR work?

PACE OneStep RT-PCR Master Mix is designed for highly specific and sensitive one-step reverse transcriptase (RT) fluorescently-reporting endpoint PCR using RNA template.

PACE OneStep RT-PCR Master Mix includes optimised components which allow reverse transcription and subsequent PCR amplification to take place in the same reaction well. 

The PACE OneStep RT-PCR reaction is comprised of three parts:

  • Assay Mix (unlabelled, forward, and reverse primers designed to amplify the target region)
  • Reverse transcriptase enzyme
  • PACE OneStep RT-PCR master Mix containing all components required for PCR amplification (post reverse-transcription) and the generation of fluorescent signals.

When combined with template RNA, these components create a PACE OneStep RT-PCR.

What is One-step RT-PCR?

A one-step RT-PCR (Reverse Transcription Polymerase Chain Reaction) allows for the amplification of a target DNA sequence directly from an RNA sample. It combines reverse transcription of RNA into complementary DNA (cDNA) and subsequent amplification of the target DNA sequence in a single reaction tube. Reverse transcription and cDNA amplification can also be carried out in two separate reactions (two-step method), but the one-step approach significantly improves and streamlines the workflow and reduces the risk of contamination.

What are the advantages of using a one-step RT-PCR over a two-step RT-PCR?

One-step RT-PCR combines reverse transcription of RNA into complementary DNA (cDNA) and subsequent amplification of the target DNA sequence in a single reaction tube. Reverse transcription and cDNA amplification can also be carried out in two separate reactions (two-step method), but the one-step approach significantly improves and streamlines the workflow and reduces the risk of contamination.

Can PACE OneStep RT-PCR Master Mix be used with different PCR instruments or platforms?

PACE OneStep RT-PCR Master Mix can be used with any standard qPCR instrument, peltier or water bath-based thermal cycler.  The fluorescent data should be captured at the PCR endpoint at 40˚C or below. 

Do I need to use a specific reaction plate or reaction volume for PACE OneStep RT-PCR Master Mix?

PACE OneStep RT-PCR Master Mix can be used with any reaction plate or well volume.  It is important that PACE OneStep RT-PCR Master Mix is used at a final 1x concentration. 

Are there any specific precautions or considerations when using PACE OneStep RT-PCR Master Mix?

RNA is highly susceptible to ubiquitous RNases.  Care should be taken when handling the samples.  An empirical optimisation of RNA concentration should be carried out by testing a sample dilution range.

If RNA samples contain EDTA, the concentration at the final reaction concentration should be no higher than 0.1 mM.

How does ProbeSure OneStep RT-PCR work?

ProbeSure OneStep RT-PCR Master Mix is designed for highly specific and sensitive one-step reverse transcriptase (RT) fluorescently-reporting PCR using RNA template.

The master mix includes optimised components which allow reverse transcription and subsequent PCR amplification to take place in the same reaction well. 

ProbeSure OneStep RT-PCR Master Mix may be used in endpoint or real-time modes.

The ProbeSure OneStep RT-PCR Master Mix reaction is comprised of two parts:

  • Assay mix comprising forward and reverse primers, and fluorogenic hydrolysis probes.
  • Universal ProbeSure OneStep RT-PCR Master Mix containing all the components required for reverse transcription and subsequent PCR.

When combined with template RNA, these components create a ProbeSure OneStep RT-PCR.

ProbeSure OneStep RT-PCR Master Mix is designed for use in PCR genotyping applications with hydrolysis probe-based assays.  It can be used with a variety of fluorogenic probe types including TaqMan®, ZEN™ probes and BHQ® / BHQplus® probes. 

Can ProbeSure OneStep RT-PCR Master Mix be used with different PCR instruments or platforms?

ProbeSure OneStep RT-PCR Master Mix can be used with any standard qPCR instrument, peltier or water bath-based thermal cycler.  The fluorescent data should be captured at the PCR endpoint at 40˚C or below. 

Do I need to use a specific reaction plate or reaction volume for ProbeSure OneStep RT-PCR Master Mix?

ProbeSure OneStep RT-PCR Master Mix can be used with any reaction plate or well volume.  It is important that ProbeSure OneStep RT-PCR Master Mix is used at a final 1x concentration. 

Are there any specific precautions or considerations when using ProbeSure OneStep RT-PCR Master Mix?

RNA is highly susceptible to ubiquitous RNases.  Care should be taken when handling the samples. An empirical optimisation of RNA concentration should be carried out by testing a sample dilution range. If RNA samples contain EDTA, the concentration at the final reaction concentration should be no higher than 0.1 mM.

Can the PACE and ProbeSure chemistries be used in digital PCR or in microfluidic systems?

We have done no work as yet with these chemistries in digital or other microfluidic systems and it is possible that a small buffer reformulation might be necessary for some platforms. If this is of particular interest to you, please contact us to discuss.

Are PACE and ProbeSure Genotyping Master Mixes compatible with Nexar and IntelliQube® platforms?

Yes, PACE and ProbeSure can be used directly on both these and many other platforms.

Legal Information
KASP™ is a trademark of LGC Biosearch Technologies
Amplifluor® is a registered trademark of Merck KGaA

still Have questions? don’t hesitate to Send us a message.

Send us a message:

This field is for validation purposes and should be left unchanged.
[fibosearch]
Name(Required)
This field is for validation purposes and should be left unchanged.

Contact us

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.