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.

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 we would suggest 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.

It is good practice to store DNA solutions in a buffer containing EDTA to prevent damage from nucleases, however, we generally recommend that the final EDTA concentration is low (0.1 mM). In any case, always 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 both PACE assay designs and KASP™ assays?

Yes. Both assays incorporate the same tail sequence and are compatible with PACE Genotyping Master Mix.

Where do I get PACE assay designs from?

We will design them for you free of charge but at present we don’t carry out laboratory validation. Simply order the primers from your oligonucleotide supplier.

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, ProbeSure Genotyping Master Mix should only be used with 5′ nuclease dependent probe based assays only. Similarly, 5′ nuclease assays cannot be used with PACE Genotyping Master Mix.

Reaction mixes

My product arrived defrosted, can I still use it?

Both PACE and ProbeSure Genotyping Master Mixes are stable for one week 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 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.

Some customers would be adding a very small volume of ROX to a master mix, i.e. if they were just going to do a small amount of genotyping. Pipetting very small volumes accurately is much more prone to error; using low ROX would make it 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.

Is PACE Genotyping Master Mix the same as LGC’s KASP™ Master Mix?

No. The PACE technology uses a completely different signal generating technology to KASP, however, despite this, PACE Genotyping Master Mix will work perfectly with KASP assays.

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

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

That said, we do have plans to release real-time optimised versions of both chemistries so watch this space!

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 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. It is 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 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.

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.

If I invest time in running my lab with 3CR Bioscience products, will you increase your prices in the future?

The aim of our company is to be the lowest priced manufacturer of the reagent types we supply. We will endeavour to reduce our prices in the future, however some small increases in prices are conceivable over the medium-to-long-term to deal with commercial realities such as suppliers’ price increases. We make the promise that we will always strive to be the most cost-effective solution for our customers. Our mission is to provide our customers with the best prices and best service in the industry and we don’t take this lightly.

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