FAQs

FAQsPost Type

How much of my primers do I have to use?

The recommended concentration of primers in the TwistAmp® Basic reaction is 480nM each. In the TwistAmp® exo, TwistAmp® fpg, and Twist Amp® nfo kits the recommended concentration of primer is 420nM each. However, the performance of some primer pairs can be improved by slightly varying their amounts in the reaction and a titration strategy (from 200nM to 600nM each) can […]

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How might I improve reproducibility of amplification curves when using TwistAmp® Liquid kits?

Reproducibility of replicate reactions can be optimised by considering the following: For low copy numbers of template, variable amplification could be because the limit of detection for an assay is being reached or because of differential agitation of reactions (with poorly agitated reactions not amplifying as well). Alternatively, the recombinase and ancillary protein in the 20x Core Reaction Mix are

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How important is it to add the TwistAmp® Liquid reaction components in the suggested order?

We recommend two possible variant approaches to making master mixes: One for when trying different oligo combinations, and one for testing different templates once the oligonucleotides are chosen. Changing the order of addition for these reactions can lead to sub-optimal RPA reactions. If using the TwistAmp® Liquid exo kit, it is best to keep the 50x Exo reagent separate from

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How do I select a probe?

If using one of the TwistAmp® kits suitable for probe use, follow the guidelines described in the assay design manual for the design of detection probes. Note that there are some sequence restrictions in choosing the position of probes within a target using the preferred TwistAmp® exo Probe system. If this presents unreasonable limitations TwistDx can help you design alternative

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How do I reconstitute a lyophilised probe?

Please follow the oligonucleotide manufacturer’s instructions for the reconstitution and storage of the probes. Typically the tube containing the lyophilised oligonucleotide will be spun briefly to collect the DNA at the bottom of the tube and an appropriate volume of T0.1E buffer (10mM Tris-HCI pH 8, 0.1mM EDTA) will be added to prepare a stock solution of 100μM. Allow the

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How can reproducibility issues be avoided when utilising small TwistAmp® Liquid reaction volumes?

Reproducibility can obviously be a problem when pipetting small volumes of liquid sub-components as variable component ratios could arise. It is advisable to make a master mix to include the Core Reaction Mix, E-mix etc. which should make things more reproducible. If it’s not feasible to make a master mix, then reverse pipetting can also improve reproducibility.

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