FAQs

Frequently asked questions

Here are some questions answered along with some tips and tricks we’ve picked up over the years that might help you. Please feel free to contact us with more questions and we’ll do our best to answer them.

Why use PACE™ over other competitive genotyping chemistries?

We believe, that PACE™ Genotyping Master Mix, is the most competitively-priced genotyping master mix for end-point, singleplex reactions.

We also believe that PACE™ suffers from reduced no template control (NTC) amplification for the following reasons:

1.  Lower magnesium level than alternatives
2.  Very thorough enzyme inactivation
3.  The structure of the reporting mechanism is such that non-specific amplification is avoided

Template considerations and DNA quality

How should DNA be prepared for genotyping?

Purification of DNA will generally lead to better genotyping data quality than using a crude extract, but it is also considerably more expensive.  If the sample number is small and the application is not especially cost constrained, we would always recommend purification.  

However, for larger scale, more cost driven applications it is common to use crude extraction methods such as hotSHOT.  Crude methods are likely to require more initial upstream testing to establish an appropriate dilution ‘window’ but once this is done the cost savings, both in terms of materials and reduced FTE, can be considerable.

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 mixes, PACE-IR™and ProbeSure-IR™. 

It is prudent to store DNA solutions in a buffer containing EDTA to prevent damage from nucleases, however we generally recommend that the final EDTA concentration at the point of use is low (0.1 mM).

Regardless of the extraction and storage methods used, it is always advisable to test the DNA first to choose the optimal dilution.

Do I need to use controls?

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 SNP / indel.

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.

Assays

What types of probes should I use with 3CR 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 Thermo Fisher Scientific’s TaqMan® MGB, IDT’s ZEN™ probes and LGC’s 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. All of these assays are compatible with PACE™ Genotyping Master Mix.

Do I have to buy PACE assays from 3CR?

No, we offer a free assay design service that enables you to order primers and prepare assays yourself.  Please see our Quick Reference Guide for making assays:

Tip: there are a few oligo manufacturers whose oligos do not work as well as others for allele-specific genotyping applications. We would strongly recommend IDT.

Can I use PACE assay designs with ProbeSure Genotyping Master Mix?

No, ProbeSureGenotyping Master Mix should be used with 5′nuclease dependent probe-based assays only.  Similarly, 5′nuclease assays cannot be used with PACEGenotyping Master Mix.

Reaction mixes

What are the optimal storage conditions for PACE master mix?

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

What is the shelf life of PACE Genotyping Master mix?

PACE Genotyping master is stable for up to two weeks at 4°C and one year when stored at -20°C.

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 these defrosted but the contents are still cold, the reagents should still be absolutely fine to use. Feel free to contact 3CR Bioscience if there are any quality issues.

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 with high ROX (500nM), low ROX (25nM) and standard ROX (150nM), whereas ProbeSure Genotyping Master Mix is available with high ROX (500nM), low ROX (25nM) and no ROX.

If you are unsure about which ROX variant is required for your qPCR machine or plate reader, please contact 3CR Bioscience and we will advise you.

Why don’t you supply ROX separately? That way you could just supply one version of PACE and ProbeSure and the customer could just add ROX themselves.

Adding a very small volume of ROX to a master mix prior to a genotyping experiment is prone to error and trying to make a mix to low ROX would make the problem worse.

Can I dilute reaction mixes to make them go further?

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

Can the PACE and ProbeSure chemistries be used for real-time PCR?

Yes and no. Both chemistries will certainly function in real-time but we have not optimised them for this purpose. You are free to test them for real-time applications (and we are happy to hear your feedback) but we cannot support their use in this way at present.

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, let us know and we might be able to work with you on this.

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.

Reaction volumes

What reaction volume should I use?

This depends on a number of factors:

  • Plate type and well geometry. If the well is very large, as is the case in a 96-well plate, larger reaction volumes should be used otherwise a significant portion of the reaction liquid volume will evaporate into the vessel’s head space. Conversely, a 1536-well plate or LGC Douglas Scientific Array Tape® has a very small well and can be used with reaction volumes below 1 µL.
  • Plate seal. Glue-backed seals can interfere with the fluorescent signal transmission. As a result, larger reaction volumes can be required to ensure that sufficient signal is detected.
  • Sample dilution. When working with crudely-extracted DNA samples a situation can arise wherein the sample requires significant dilution to titrate the PCR-inhibitors down to a level that allows PCR to proceed. Dilution of crude samples keeps costs down significantly and is commonly used in high-throughput scenarios (see section on DNA sample preparation). The issue with this however is that, in some cases, the dilution required concomitantly reduces the DNA concentration to a level that is too low for efficient PCR. If this is the case, we would recommend use of our inhibitor-resistant master mixes, PACE-IR™and ProbeSure-IR™. It is also sometimes feasible however to increase the volume of the reaction thus using its own volume to provide part of the dilution.
  • Economics. As long as you have the right plates and equipment, smaller reaction volumes mean you use less master mix per genotype.

Thermal cycling

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 the cycling conditions for different assays?

The very large majority of assays will work well with the standard cycling conditions. There are some exceptions however, such as assays in areas of very high GC content or where homology is an issue. If you experience any issues with cycling conditions for difficult assays, please contact us for support

Fluorescent readers

Gain settings

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 ‘max out’ the reader leading to nonsense results, which are not always easy to spot and can therefore lead to incorrect data. We advise running a first test with the reader set for optimised gain, and then use the values suggested by the reader.

Pricing and legal

Why are 3CR Bioscience’s prices so competitive?

We want our customers to be able to do more work, to generate more data and by definition do things that you could not previously have done. We fundamentally believe that this situation, along with our eagerness to work with you to solve problems and even develop new products, leads to a partnership where everybody benefits.

Why can I not find a schema showing how the PACE chemistry works?

The reason is simply that we have not finished patenting the PACE technology so cannot divulge how it works.

Working with customers

Does 3CR Bioscience work with customers to develop products?

Yes!

We are very interested in talking to customers both to develop new products and to develop existing products into new areas.

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

As long as you will order this pack size regularly then, yes, this is possible.

Bespoke products

3CR Bioscience are always interested in developing bespoke products for customers so, providing you are going to order enough of the product, we are happy to make it for you. Please contact us to discuss