PACE 2.0 Genotyping Master Mix uses a novel, universal, fluorescent reporting cassette to produce machine-readable fluorescent signals corresponding to genotypes. PACE 2.0 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 2.0 Genotyping Master Mix is supplied 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 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 2.0 Genotyping Master Mix.
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.
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.
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.
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.
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.
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.
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.