The RPA product (10 µL) was transfered to 40 µL of the CRISPR-Cas12a reaction mixture containing 100 nM crRNA (Sangon Biotech, Shanghai, China), 50 nM Cas12a (NEB, Ipswich, UK) and 250 nM ssDNA reporter (Sangon Biotech, Shanghai, China). Then, the reactions (50 µL in a PCR tube) were incubated in a fluorescence plate reader (Molecular Devices, California, USA) or a Real Time PCR Detection System (Bio-Rad, Watford, UK) for up to 60 min at 37 °C with fluorescent signals collected every 30 s (ssDNA FQ substrates = λex: 485 nm; λem: 535 nm).
Crrna
Manufactured by Sangon
Sourced in China
CrRNA is a type of RNA used in CRISPR gene editing technology. It serves as a guide for the Cas9 enzyme to locate and cleave specific DNA sequences.
Automatically generated - may contain errors
Lab products found in correlation
Qiaamp dna mini kit, by Qiagen (2 mentions)
Rpa primers, by Sangon (2 mentions)
Real time pcr detection system, by Bio-Rad (1 mentions)
Cas12a, by New England Biolabs (1 mentions)
Ssdna reporter, by Sangon (1 mentions)
Fluorescence plate reader, by Molecular Devices (1 mentions)
Engen lba cas12a cpf1, by New England Biolabs (1 mentions)
100 bp dna ladder, by Transgene (1 mentions)
Nebuffer r2, by New England Biolabs (1 mentions)
Twistamp exo kit, by Twist Bioscience (1 mentions)
Twistamp basic kit, by Twist Bioscience (1 mentions)
Nebuffer 3, by New England Biolabs (1 mentions)
Lbcas12a protein, by New England Biolabs (1 mentions)
Rna inhibitor, by New England Biolabs (1 mentions)
Dna extraction kit, by Tiangen Biotech (1 mentions)
Lbcas12a, by New England Biolabs (1 mentions)
6 protocols using crrna
1
CRISPR-Cas12a Fluorescence Assay
2
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).
3
Rapid Detection of Aphelenchoides Nematodes
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The nematodes used in this study are listed in Table 1 . Two rice grain samples, AB1 and AB2, infected by A. besseyi were collected from paddy fields in Shanghai and Nanjing, respectively. The related Aphelenchoides spp., A. fragariae (AF), and A. subtenuis (AS), were intercepted in Shanghai ports and saved in Shanghai Entry-Exit Inspection and Quarantine Bureau. M. incognita (MI) were kindly provided by Prof. Xuan Wang (Nanjing Agricultural University, Nanjing, China) (Wang et al., 2018 (link)).
RPA primers, crRNA, and ssDNA reporters were synthesized by Sangon Biotech (Shanghai, China). The RPA assay kit (TwistAmp Basic kit, TABAS03KIT) was purchased from TwistDx Ltd. (TwistDx Ltd., United Kingdom). The LbCas12a (cpf1) nuclease (32108-03) was purchased from Tolo Biotech (Tolo Biotech, Anhui, China). The lateral flow strip (JY0301) was purchased from Warbio Biotech (Nanjing, China).
RPA primers, crRNA, and ssDNA reporters were synthesized by Sangon Biotech (Shanghai, China). The RPA assay kit (TwistAmp Basic kit, TABAS03KIT) was purchased from TwistDx Ltd. (TwistDx Ltd., United Kingdom). The LbCas12a (cpf1) nuclease (32108-03) was purchased from Tolo Biotech (Tolo Biotech, Anhui, China). The lateral flow strip (JY0301) was purchased from Warbio Biotech (Nanjing, China).
4
Rapid Detection of Biothreat Agents
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The following bacterial strains were available in our lab: F. tularensis, Brucella melitensis, Brucella abortus, Burkholderia pseudomallei, Burkholderia mallei, Bacillus anthracis, Staphylococcus aureus, Bacillus thuringiensis, Yersinia pestis, Salmonella typhi, Bacillus subtilis, Escherichia coli, Vibrio vulnificus, Staphylococcus epidermidis, Vibrio parahaemolyticus, Bacillus cereus, and Vibrio cholerae. RPA primers, RPA probes, crRNA, and fluorescent single-stranded DNA reporter (Flu-ssDNA) were synthesized by Shanghai Sangon Biotech Co., Ltd. (China). LbCas12a protein, NEBuffer 3.1, and RNA inhibitor were provided by New England BioLabs, Inc. (USA). TwistAmp™ Exo Kit and TwistAmp™ basic Kit were provided by TwistDx (Cambridge, UK). A positive reference plasmid for F. tularensis detection (pEASY-T1-TUL4) was constructed by our lab. DNase/RNase-free distilled, deionized water (DDH2O) was provided by Tiangen Biochemical Co., Ltd. A QIAamp™ DNA Mini Kit (Qiagen, Germany) was used to extract bacterial strain genomes, followed by the user manual's protocol.
5
CRISPR-Cas12a-based Lateral Flow Assay
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Lateral flow assay reaction system was similar to that of DETECTR assay except for the 5′-FAM-ssDNA (TTATT)-Biotin-3′ reporter (Sangon, China). 4ul of the RAA product was added into the CRISPR/Cas12a reaction mixture, containing 2 μl NEBuffer 2.1 (10 x), 2 μl LtCas12a (10 uM), 4 μl crRNA (10 uM), 0.7μl ssDNA reporter (5′-FAM-TTATT-Biotin-3′) (Sangon, China) (10 μM), and 7.3 μl nuclease-free water. The above mixture was incubated at 37°C for 30–50 min. The 20 μl reaction volume was finally added with 80ul strip buffer and then loaded onto the strips (Milenia Biotec GmbH, German). Photograph and record the result after incubating at room temperature for 5 min. Gray scale values of bands were read by ImageJ software.
6
CRISPR/Cas12a-based Rapid Pathogen Detection
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All the DNA strands (Table S1 ) were synthesized by Sangon Biotech Co., Ltd (Shanghai, China). LbCas12a and RNA enzyme inhibitors were purchased from NEB UK, DNA extraction kit were from Tiangen (Tianjin, China), recombinase polymerase amplification (RPA) kit of TwistAmp ®Basic and qPCR kit were purchased from Vazyme (Nanjing, China). The structure and sequence of crRNA were designed and screened on a professional website named CHOPCHOP (http://chopchop.cbu.uib.no/ ). crRNA was ordered from Sangon Biotech Co., Ltd (Shanghai, China). The feasibility verification of crRNA was performed via the process that the CRISPR/Cas12a system provides a trans-cleavage for foreign ssDNA-FQ to release a fluorescence signal. The lateral flow strips integrated with colloidal gold probe, capture antibody and streptavidin were purchased from Lesunbio Co. Ltd. (Wuxi, China).All bacterial strains (listed in Table S2 ) were isolated and maintained in our laboratory. All isolates were verified by PCR and 16S rRNA gene sequencing.
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