Twistamp basic rpa kit

Manufactured by Twist Bioscience

Sourced in United Kingdom

The TwistAmp Basic RPA kit is a reagent system designed for rapid isothermal amplification of DNA. The kit contains all the necessary components for performing recombinase polymerase amplification (RPA) reactions.

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Lab products found in correlation

Nanodrop 2000c, by Thermo Fisher Scientific (2 mentions) Qubit dsdna hs assay kit, by Thermo Fisher Scientific (2 mentions) Twistamp basic rt rpa kit, by Twist Bioscience (1 mentions) M mulv reverse transcriptase, by New England Biolabs (1 mentions) Qiacube robotic workstation, by Qiagen (1 mentions) Thermomixer, by Eppendorf (1 mentions) 100 bp dna ladder, by Qiagen (1 mentions) Pcr cleanup kit, by Qiagen (1 mentions)

Spelling variants of this product

TwistAmp Basic kit (88 citations) TwistAmp (6 citations) TwistAmp Basic (5 citations) Basic RPA kit (2 citations) TwistAmp Basic RT-RPA kit (2 citations)

5 protocols using twistamp basic rpa kit

Rapid Isothermal DNA Amplification

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Fast isothermal amplification was performed using
the TwistAmp Basic RPA kit (TwistDx, U.K.). Reaction mixtures (12.5
μL) were prepared with rehydrated buffer, 14 mM magnesium acetate,
200 nM each forward and reverse primer, 4 ng of gDNA, and the enzyme
pellet. Solutions were incubated at 37 °C for 10 min in an oven
(Beschickung—Loading Model 100-800, Germany).

Lázaro A., Maquieira Á, & Tortajada-Genaro L.A. (2022). Discrimination of Single-Nucleotide Variants Based on an Allele-Specific Hybridization Chain Reaction and Smartphone Detection. ACS Sensors, 7(3), 758-765.

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Rapid RNA detection via RT-RPA

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For cell line experiments, the TwistAmp Basic RPA kit (Twist-DX, UK) was used with slight modifications to manufacturer’s instructions. Briefly, 1 μl of extracted RNA, 50 units of MMuLV reverse transcriptase (New England Biolabs, UK) and 500 nM of each primer (Table 1) were added to make a 12.5 μl reaction volume prior to incubation at 39 °C for 20 min. For all other experiments, the TwistAmp Basic RT-RPA kit (Twist-DX, UK) was used with slight modifications to manufacturer’s instructions to simultaneously generate cDNA and rapidly amplify the cDNA templates in a single tube reaction. Briefly, 2 μl of extracted RNA and 500 nM of each primer (Table 1) were added to make a 12.5 μl reaction volume prior to incubation at 41 °C for 30 min.

Koo K.M., Wee E.J., Mainwaring P.N, & Trau M. (2016). A simple, rapid, low-cost technique for naked-eye detection of urine-isolated TMPRSS2:ERG gene fusion RNA. Scientific Reports, 6, 30722.

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Rapid Nucleic Acid Amplification via RPA

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Nucleic acid amplification was performed with the TwistAmp Basic RPA Kit (TwistDX, cat# TABAS01kit) as recommended by the manufacturer with some modifications. 12.5 μL reactions were performed at 38 °C for 20 min using 1 μL of the nucleic acid extraction and 800 nM of each primer (Table 1). Following amplification, 3 μL of the RPA reaction was visualized by gel electrophoresis.

Ng B.Y., Wee E.J., West N.P, & Trau M. (2015). Rapid DNA detection of Mycobacterium tuberculosis-towards single cell sensitivity in point-of-care diagnosis. Scientific Reports, 5, 15027.

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Optimized isothermal DNA amplification

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For the optimisation assays, two cell cultures were used, cell line SK-N-AS (ATCC, wildtype for KRAS codon 12) and cell line HCT 116 (ATCC, c.38G>A), from a primary carcinoma tissue. For method validation, healthy and oncological patients were recruited for the present study according to ethics regulations. QIAcube robotic workstation (Qiagen, Germany) extracted genomic DNA from 5-μm thick formalin-fixed paraffinembedded tissue sections. The used reagents were mini kit FFPE Qiagen. The concentration of the DNA extracts (ng μL -1 ) was obtained by spectrophotometry using NanoDrop 2000c (ThermoFisher Sci., USA), and by fluorimetry using Qubit dsDNA HS Assay Kit (ThermoFisher Sci.).
The reagents used for the genomic DNA amplification came in the TwistAmp Basic RPA kit (TwistDx, UK). The mixtures (50 μL) for the blocked isothermal amplification were prepared with the enzyme pellet in rehydrated buffer, 480 nM of magnesium acetate, 480 nM of upstream primer and downstream primer, 70 nM of blocking agent, 4 ng of genomic DNA, 0.01 mM aminoally-dUTP-Cy5. The employed heating system was an Eppendorf Thermomixer with MTP adapter (300 rpm, Eppendorf), operating at 37 °C for 40 min. Also, real time thermocycler (TS2, Qiagen) was used for the optimisation assay.
The reaction mechanism is described in Fig. SI.1.

Martorell S., Tortajada-Genaro L.A, & Maquieira Á. (2019). Magnetic concentration of allele-specific products from recombinase polymerase amplification. Analytica chimica acta, 1092.

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Rapid Isothermal Amplification Protocol

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RPA was performed using the TwistAmp Basic RPA kit (TwistDx). RPA reaction mix was assembled by combining 29.5 µL TwistAmp Basic Rehydration buffer, 0.5 µM of each primer, 5 µL template and water in a total volume of 47.5 µL. Primers were designed using PrimedRPA 37 . 5'-overhangs included in reverse primers were added manually to the primers provided in PrimedRPA output. The TwistAmp Basic pellet was rehydrated by adding 47.5 µL RPA reaction mix and was incubated for 5 minutes at room temperature until completely dissolved. The reaction mix was mixed by pipetting and divided into 19 µL aliquots. To start the reaction, 1 µL of MgOAc (provided with TwistAmp Basic kit) was added. The reaction was incubated at 42 ºC for 20 minutes. The reaction was terminated by PCR clean-up or by quenching the reaction on ice. After a routine clean-up using a PCR clean-up kit (QIAGEN), amplification was confirmed by agarose gel electrophoresis using a 2% agarose gel and a GeneRuler 100 bp DNA Ladder.

Brink M.v.d., Tandar S.T., Akker T.A.P.v.d., Jovikj S., Defourt V., Langelaar T.G.B., Delzenne T.O., Strien K.v., Schonk A.W., Beers A., Golov E., Chong L., Bergman G.Ö., Meijdam J.J.W.D., Crooijmans M., Claessen D., & Winde J.H.d. (2020). Rapidemic, a versatile and label-free DNAzyme-based platform for visual nucleic acid detection.

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