All oligonucleotides (Table S1 ) used in this study were purchased from Bionics (Seoul, Republic of Korea) and Integrated DNA Technologies (Coralville, IA, USA). T7 RNA polymerase, T7 RNA polymerase buffer, and RNase inhibitors were obtained from Enzynomics (Daejeon, Republic of Korea). The HiScribe T7 High Yield RNA synthesis kit, Monarch RNA cleanup kit, DNase I, DNase I reaction buffer, NEBuffer™ r2.1, and rNTP were purchased from New England Biolabs (Ipswich, MA, USA). Plates with 384 black wells were purchased from SPL Life Sciences (Pocheon, Republic of Korea). To1-3PEG-Biotin Fluorophore (To1-biotin) was obtained from Applied Biological Materials (Richmond, BC, Canada). LbuCas13a was purchased from SignalChem Diagnostics (St. Louis, MO, USA).
Nebuffer r2
Manufactured by New England Biolabs
Sourced in United States, China
NEBuffer™ r2.1 is a ready-to-use buffer solution designed for use in various molecular biology applications. It provides an optimal environment for the activity of various enzymes, including restriction enzymes, DNA modifying enzymes, and other DNA-manipulating proteins. The buffer composition is designed to maintain the appropriate pH, ionic strength, and cofactor concentrations to ensure efficient and reliable performance of these enzymes.
Automatically generated - may contain errors
Lab products found in correlation
Engen lba cas12a cpf1, by New England Biolabs (2 mentions)
Dnase 1, by New England Biolabs (1 mentions)
T7 rna polymerase, by Enzynomics (1 mentions)
Monarch rna cleanup kit, by New England Biolabs (1 mentions)
Dnase 1 reaction buffer, by New England Biolabs (1 mentions)
Hiscribe t7 high yield rna synthesis kit, by New England Biolabs (1 mentions)
Crrna, by Integrated DNA Technologies (1 mentions)
Rpa primers, by Integrated DNA Technologies (1 mentions)
Cytation 5 imaging reader, by Agilent Technologies (1 mentions)
Ascas12a, by Integrated DNA Technologies (1 mentions)
Twistamp basic kit, by Twist Bioscience (1 mentions)
Crrna, by Sangon (1 mentions)
100 bp dna ladder, by Transgene (1 mentions)
Qiaamp dna mini kit, by Qiagen (1 mentions)
Engen lba cas12a, by New England Biolabs (1 mentions)
Spectramax id3 microplate reader, by Molecular Devices (1 mentions)
Glycine, by Fujifilm (1 mentions)
Sucrose, by Fujifilm (1 mentions)
Dextran, by Fujifilm (1 mentions)
Trehalose dihydrate, by Fujifilm (1 mentions)
7 protocols using nebuffer r2
1
CRISPR-Cas13a-based Biomolecular Assay
2
Rapid CRISPR-Cas12a Detection Assay
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TwistAmp@ Basic kit was purchased from TwistDx™. The RPA primers, crRNA, AsCas12a, and fluorophore-quencher probes were all obtained from Integrated DNA Technologies, and detailed information about the synthetic oligonucleotides are listed in Table 2 . The RPA primer sets were designed using PrimerQuest™ Tool. Additionally, NEBuffer™ r2.1 was purchased from New England Biolabs. The RPA reaction was conducted based on the instructions: A mixture of 29.5 μL of rehydration buffer, 11.2 μL of nuclease-free water, and 2.4 μL each of forward and reverse primers (10 μM) was added to the enzyme pellet. Then, 2 μL of purified DNA and 2.5 μL of MgOAc (280 mM) were added and mixed to achieve a total volume of 50 μL. The mixture was incubated at 37 °C for 20 min. Following the incubation, 2 μL of RPA amplicons were added to a pre-assembled CRISPR-Cas12a mixture comprising 50 nM of AsCas12a, 62.5 nM of crRNA , 10× buffer, and 2.5 μM of ssDNA-FQ probe, resulting in a final reaction volume of 20 μL. The reaction solution was incubated at 37 °C for 30 min. After the incubation, the mixture was excited by a blue light transilluminator (brand: SmartBlue, Part number: NEB-E4100, excitation wavelength of 465 nm) for naked-eye observation. Finally, 20 μL of nuclease-free water was added to 5 μL of the mixture, which was then characterized by an Agilent BioTek Cytation 5 imaging reader.
3
Isothermal Amplification with RPA-Cas12a
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Recombinase polymerase amplification Kits used for isothermal amplification were purchased from Lesunbio Co., Ltd. (Wuxi, China). Primers for RPA reaction, crRNA and double distilled water were ordered from Sangon Biotech Co., Ltd. (Shanghai, China). The ssDNA was synthesized by TianyiHuiyuan Biotechnology Co., Ltd. (Beijing, China). The 100 bp DNA ladder was obtained from TransGene Biotech Co., Ltd. (Beijing, China). EnGen® Lba Cas12a (Cpf1) and NEBuffer™ r2.1 were purchased from New England BioLabs (Beijing, China). QIAamp DNA Mini Kits used for DNA extraction were obtained from Qiagen GmbH (Hilden, Germany).
4
Cas12a-Mediated Lateral Flow Assay
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Two crRNAs were designed using the CRISPOR web tool (http://crispor.org ) [28 (link)] to target the RPA amplicons. For LFA readout, single-stranded DNA (ssDNA) was labeled with the fluorophores 6-carboxyfluorescein (FAM) and biotin, as indicated in Table 1 . For fluorescence detection, the ssDNA was labeled with FAM and Black Hole Quencher1 (BHQ1). The crRNAs and the ssDNAs were synthesized by Bioneer (Korea).
To detect Cas12a-mediated trans-cleavage activity using the LFA, 20 μL of the Cas12a reaction mixture was prepared with EnGen® Lba Cas12a (1 μM, M0653T, NEB, USA), NEBuffer r2.1 (B6002S, NEB, USA), crRNA (1 μM), RPA products, labeled ssDNA (10 μM), and distilled water. The mixture was incubated at 37 °C for 30 min using the ProFlex PCR System. After incubation, 20 μL of the reaction mixture was mixed with 100 μL of assay buffer of the HybriDetect – Universal Lateral Flow Assay Kit (MGHD1, Milenia Biotec, Germany) in a 1.5 mL tube.
To quantify the fluorescence, 100 μL of Cas12a reaction mixture was prepared and performed in a 96-well black polystyrene microplate (SPL, Korea). The fluorescence was detected and analyzed with the SpectraMax iD3 microplate reader (Molecular Devices, USA) at excitation and emission wavelengths of 470 nm and 520 nm, respectively. Emission was read in relative fluorescence units (RFU) to compare samples within each experiment.
To detect Cas12a-mediated trans-cleavage activity using the LFA, 20 μL of the Cas12a reaction mixture was prepared with EnGen® Lba Cas12a (1 μM, M0653T, NEB, USA), NEBuffer r2.1 (B6002S, NEB, USA), crRNA (1 μM), RPA products, labeled ssDNA (10 μM), and distilled water. The mixture was incubated at 37 °C for 30 min using the ProFlex PCR System. After incubation, 20 μL of the reaction mixture was mixed with 100 μL of assay buffer of the HybriDetect – Universal Lateral Flow Assay Kit (MGHD1, Milenia Biotec, Germany) in a 1.5 mL tube.
To quantify the fluorescence, 100 μL of Cas12a reaction mixture was prepared and performed in a 96-well black polystyrene microplate (SPL, Korea). The fluorescence was detected and analyzed with the SpectraMax iD3 microplate reader (Molecular Devices, USA) at excitation and emission wavelengths of 470 nm and 520 nm, respectively. Emission was read in relative fluorescence units (RFU) to compare samples within each experiment.
5
Cas12a-based Nucleic Acid Detection
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All nucleic acids used in this work including target dsDNA (tgDNA), crRNA, and FQ reporter were purchased from Integrated DNA Technologies (Singapore). The corresponding sequences are listed in Table S1. † NEbuffer r2.1 and Lba Cas12a were bought from New England Biolabs (Ipswich, MA, USA). Filter papers (Whatman Grade 1 (WF1) and Grade 541 (WF541)) were purchased from GE Healthcare Life Sciences (Marlborough, MA, USA), while Advantec 5C (A5C) was obtained from Toyo Roshi, Co., Ltd (Tokyo, Japan). Bovine serum albumin (BSA), trehalose dihydrate, sucrose, glycine, and dextran (MW: 70 000) were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Nuclease-free water was purchased from Nacalai Tesque (Kyoto, Japan).
6
Isothermal Amplification Using Recombinase Polymerase
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Recombinase polymerase–based amplification kit for isothermal amplification was purchased from Msunflowers Biotech Co., Ltd. (Beijing, China). The 100-bp DNA marker and the EasyPure® Genomic DNA Kit for genomic DNA extraction and purification were obtained from TransGene Biotech Co., Ltd. (Beijing, China). EnGen® Lba Cas12a (Cpf1) and NEBuffer r2.1 were purchased from New England Biolabs (Beijing, China). The ABI 7500 FAST real-time PCR platform (Applied Biosystems, USA) was used as the fluorescence reader. An imaging system (Gel Doc XR C, Bio-Rad, USA) was used for gel image taken.
7
Rapid CRISPR-based Pathogen Detection
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Primers for RPA were synthesized by TsingKe Biological Technology (Beijing, China). Recombinant RNase inhibitor, crRNA, and RNase-free water were obtained from Takara Bio (Beijing, China). The ssDNA reporter was synthesized by Sangon Biotech (Shanghai, China). The TIANamp Bacteria DNA kit was purchased from TIANGEN (Beijing, China). The TwistAmp Basic Kit for RPA reaction was purchased from TwistDx (Cambridge, UK). Lba Cas12a (Cpf1) and NEBuffer r2.1 were purchased from New England Biolabs (Beijing, China). Milenia HybriDetect for lateral flow detection was purchased from Milenia Biotec (Gießen, Germany).
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