The CRISPR-Cas12a detection mixture was modified from the previous protocols (19 (link), 32 (link), 42 (link)). Two different mixtures (low and high concentration) of Cas12a and crRNA were used for crRNA screening and CRISPR-Cas12a detection. The low concentration contained 1× NEB buffer 2.1, 40 nM Cas12a (NEB), 40 nM crRNA, and water to 17.6 μL (10% excess). The high concentration contained 1× NEB buffer 2.1, 100 nM Cas12a (NEB), 200 nM crRNA, and water to 17.6 μL (10% excess). Both mixtures were incubated at 37°C for 30 min; after incubation, 2.2 μL (10% excess) fluorophore-quenched ssDNA fluorescent reporter (1 μM, final concentration at 100 nM,/56-FAM/TTATTATT /3BHQ1/; Beijing Genomics Institute) and 2.2 μL (10% excess) of RPA/RT-RPA products were added. The 20-μL reaction mixtures were transferred to a 384-well plate (black, flat bottom; Corning) for fluorescent intensity monitoring. Fluorescent intensities were monitored every 5 min at 37°C for 1.5 h by Synergy H1 microplate reader (BioTeK) at FAM channel (excitation/bandwidth, 484/12.5; emission/bandwidth, 530/12.5). As for the fluorescent detection in tubes, the 0.2-mL 8-strip tubes were incubated at 37°C before visualizing by ChemiDoc touch imaging system (Bio-Rad) under UV light or blue LED light with a filter, and the detection was performed at 30 min and 1.5 h, respectively (32 (link)).
Cas12a
Manufactured by New England Biolabs
Sourced in United States, United Kingdom
Cas12a is a CRISPR-associated endonuclease that recognizes and cleaves specific DNA sequences. It is a powerful tool for genome editing and can be programmed to target multiple DNA sites simultaneously.
Automatically generated - may contain errors
Lab products found in correlation
Real time pcr detection system, by Bio-Rad (4 mentions)
Ssdna reporter, by Sangon (3 mentions)
Fluorescence plate reader, by Molecular Devices (3 mentions)
Synergy h1 microplate reader, by Agilent Technologies (2 mentions)
56 fam ttattatt 3bhq1, by BGI Group (2 mentions)
Chemidoc touch imaging system, by Bio-Rad (2 mentions)
Twistamp basic powder, by New England Biolabs (2 mentions)
Protoscript 2 reverse transcriptase, by New England Biolabs (2 mentions)
Crrna, by Sangon (1 mentions)
Applied biosystems 7500 real time pcr system, by Thermo Fisher Scientific (1 mentions)
T100 pcr thermal cycler, by Bio-Rad (1 mentions)
Lightcycler 96 qpcr, by Roche (1 mentions)
Gelstain, by Transgene (1 mentions)
Depc water dnase rnase free, by Beyotime (1 mentions)
Ezup column bacterial genomic dna extraction kit, by Sangon (1 mentions)
Nuclease free water, by New England Biolabs (1 mentions)
Amv reverse transcriptase, by New England Biolabs (1 mentions)
Temed, by Bio-Rad (1 mentions)
Acrylamide bis acrylamide solution, by Bio-Rad (1 mentions)
Agarose, by Thermo Fisher Scientific (1 mentions)
16 protocols using cas12a
1
CRISPR-Cas12a Detection Assay Optimization
2
Cas12a-Based Electrochemical Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The Cas12a reaction
was carried out in
a 30 μL reaction volume containing 33.3 nM Cas12a (NEB, M0653S),
24.3 nM gRNA (IDT), 1× NEB buffer 2.1 (NEB, B7202), and variable
concentrations of DNA. For the plasmid data generated inFigure 2 , the final reaction
volumes contained 100 ng of plasmid DNA. LAMP reaction products were
diluted by a factor of 100 before inputting 3 μL of the LAMP
amplicon into the Cas12a reaction mix. Eighteen microliters of the
Cas12a reaction was deposited on the functionalized electrodes. The
electrodes were sealed in Petri dishes and placed in a 37 °C
oven for 1 h. After 1 h, the electrodes were rinsed twice with 200
μL of buffer and measured with 200 μL of buffer using
square wave voltammetry. The peak current of the methylene blue square
wave voltammograms before and after treatment with Cas12a was calculated,
and the ratio of the peak currents after treatment with Cas12a to
before treatment with Cas12a was computed.
was carried out in
a 30 μL reaction volume containing 33.3 nM Cas12a (NEB, M0653S),
24.3 nM gRNA (IDT), 1× NEB buffer 2.1 (NEB, B7202), and variable
concentrations of DNA. For the plasmid data generated in
volumes contained 100 ng of plasmid DNA. LAMP reaction products were
diluted by a factor of 100 before inputting 3 μL of the LAMP
amplicon into the Cas12a reaction mix. Eighteen microliters of the
Cas12a reaction was deposited on the functionalized electrodes. The
electrodes were sealed in Petri dishes and placed in a 37 °C
oven for 1 h. After 1 h, the electrodes were rinsed twice with 200
μL of buffer and measured with 200 μL of buffer using
square wave voltammetry. The peak current of the methylene blue square
wave voltammograms before and after treatment with Cas12a was calculated,
and the ratio of the peak currents after treatment with Cas12a to
before treatment with Cas12a was computed.
3
Rapid CRISPR-Cas12a Diagnostic Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The product of dRAA assay was used as the target of CRISPR-Cas12a system. The Cas12a trans-cleavage reaction was performed as follows: 5 μl of 1,000 nm Cas12a (M0653T; New England Biolabs Inc., MA, United States) and 5 μl of 400 nm crRNAmix (crRNA mixture), consisting of crRNA for aerA (ACR) and crRNA for hlyA (HCR), were preincubated at 37°C for 20 min to form Cas12a-crRNA complex. 10 μl of 1,000 nm ssDNA-FQ, 10 μl of 1 × NEB buffer 2.1, and 2 μl of dRAA product were added to the tube containing 10 μl of Cas12a-crRNA complex. After softly vortexed for 8 s, the tube containing 32 μl of mixture was incubated at 37°C for 35 min. The results can be read with an UV flashlight or a multifunctional microplate reader (λex: 485 nm and λem: 520 nm). In this study, we optimized the concentration of Cas12a, the concentration of ssDNA-FQ, and the Cas12a cleavage time.
4
Cas12a-based Fluorescence Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The RPA or PCR product (5 μL) was mixed with 20 μL of the 1x Cas12a reaction mixture containing 50 nM Cas12a (NEB, Ipswich, UK), 100 nM crRNA (BIOLIGO, Shanghai, China), and 250 nM ssDNA reporter (Sangon, Shanghai, China). Then, signal was collected using a fluorescence plate reader (Molecular Devices, California, USA) or a Real-Time PCR Detection System (Bio-Rad, Watford, UK) for up to 120 min at 37 °C. The fluorescence intensity data was expressed as Relative fluorescence units (RFU) or baseline subtracted RFU.
5
CRISPR-Cas12a Detection Assay Optimization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The CRISPR-Cas12a detection mixture was modified from the previous protocols (19 (link), 32 (link), 42 (link)). Two different mixtures (low and high concentration) of Cas12a and crRNA were used for crRNA screening and CRISPR-Cas12a detection. The low concentration contained 1× NEB buffer 2.1, 40 nM Cas12a (NEB), 40 nM crRNA, and water to 17.6 μL (10% excess). The high concentration contained 1× NEB buffer 2.1, 100 nM Cas12a (NEB), 200 nM crRNA, and water to 17.6 μL (10% excess). Both mixtures were incubated at 37°C for 30 min; after incubation, 2.2 μL (10% excess) fluorophore-quenched ssDNA fluorescent reporter (1 μM, final concentration at 100 nM,/56-FAM/TTATTATT /3BHQ1/; Beijing Genomics Institute) and 2.2 μL (10% excess) of RPA/RT-RPA products were added. The 20-μL reaction mixtures were transferred to a 384-well plate (black, flat bottom; Corning) for fluorescent intensity monitoring. Fluorescent intensities were monitored every 5 min at 37°C for 1.5 h by Synergy H1 microplate reader (BioTeK) at FAM channel (excitation/bandwidth, 484/12.5; emission/bandwidth, 530/12.5). As for the fluorescent detection in tubes, the 0.2-mL 8-strip tubes were incubated at 37°C before visualizing by ChemiDoc touch imaging system (Bio-Rad) under UV light or blue LED light with a filter, and the detection was performed at 30 min and 1.5 h, respectively (32 (link)).
6
Quantifying pUC57-orf068 Copy Number
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The copy number of pUC57-orf068 was calculated. The pUC57-orf068 DNA was 10-fold serially diluted from 5 × 105 to 5 × 10−1 copies/μL. PCR was performed on the series dilution template, and the PCR products were subjected to 1% agarose gel electrophoresis. Each Cas12a cleavage reaction system with a total volume of 17 μL contained 250 nM Cas12a (New England Biolabs, MA, USA), 500 nM crRNA, 1.5 µM JOE-dye single-stranded DNA-fluorophore-quencher (ssDNA-FQ) reporter, 3 μL of above PCR product, and 2 μL of NEBuffer 2.1. After the preparation of the reaction system, the reaction tubes were immediately transferred to an Applied Biosystems® 7500 real-time PCR system (Applied Biosystems, CA, USA) at 37 °C with one reading cycle per 90 s. Reverse primers containing crRNA complementary sequences were used as ssDNA activators (Table S1 ). The results were displayed as amplification curves.
7
Cas12a-Mediated LAMP Detection System
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The LAMP assay was performed according to Section 2.3 . The Cas12a-mediated detection system contained 1.95 μL 10× NEBuffer 2.1, 1.3 μL of Cas12a (1, 2, 3, and 4 μM) (New England Biolabs, Ipswich, MA, USA), 1.95 μL of crRNA (0.5, 1.0, 1.5, 2.0, and 2.5 μM), 1.3 μL of report DNA (20, 40, 60, 80, and 100 μM), and 13 μL of LAMP amplicons as an activator. Then, 19.5 μL of mixture was incubated a thermostatic metal bath at 25 °C, 31 °C, 37 °C, 43 °C, and 49 °C for 30 min. The products of LAMP cleaved by Cas12a were characterized by capillary electrophoresis.
8
Fluorescence-based Cas12a Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The RPA or PCR product (5 µL) was mixed with 20 µL of the Cas12a reaction mixture containing 50 nM Cas12a (NEB, Ipswich, UK), 100 nM crRNA (BIOLIGO, Shanghai, China), and 250 nM ssDNA reporter (Sangon, Shanghai, China). Then, signal was collected using a uorescence plate reader (Molecular Devices, California, USA) or a Real-Time PCR Detection System (Bio-Rad, Watford, UK) for up to 120 minutes at 37°C.
9
CRISPR-Cas12a Fluorescence Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
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).
10
Rapid SARS-CoV-2 Detection using RT-RPA-CRISPR
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!