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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.
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.
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.
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.
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.
Yes. PACE works very well for insertion / deletion genotyping.
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.
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.
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.
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.
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.
No, PACE Genotyping Master Mix should be used with PACE assays. Similarly, PACE assays cannot be used with ProbeSure Genotyping Master Mix.
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.
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.
Yes, PACE 2.0 Genotyping Master Mix is suitable for real-time applications.
This should not be necessary. Cycling conditions are described in detail in the PACE® Genotyping Master Mix User Guide.
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.
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.
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.
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.
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.
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 firstname.lastname@example.org and we will advise you.
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.
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.
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.
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.
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).
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.
Yes, we offer custom pathogen detection kits for this type of application. Please contact email@example.com with the relevant sequence information.
No, we do not need genome coordinates.
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.
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.
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.
Please contact firstname.lastname@example.org with the relevant sequence information. We will discuss your requirements and make the necessary assay designs.
Yes, you can. We will order IDT gBlocksTM templates on which to test your assays. There will be an additional charge for the gBlocks.
No. However, 5’ nuclease assays are available from a range of manufacturers, such as IDT.
We are happy to manufacture a custom pack size for users that are planning to make a regular order of that pack size.
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.
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.
No, these plates are not supplied with a barcode. You will need to source your barcode separately, if required.
No, our 384-well plates will warp under autoclave conditions.
Yes, we can send a sample plate for you to test. Please email your contact details to email@example.com, along with the part code of the plate.
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:
When combined with template RNA, these components create a PACE OneStep 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.
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.
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.
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.
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.
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:
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.
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.
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.
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.
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.
Yes, PACE and ProbeSure can be used directly on both these and many other platforms.