nebuffer 2 1  (New England Biolabs)


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    New England Biolabs nebuffer 2 1
    The optimization of buffer types, complex concentrations and incubation time for the trans‐cleavage activity of the LbCas12a/crRNA system. (a) Four buffers were evaluated for their effect on LbCas12a trans‐cleavage activity after 30 min of incubation with 50 n m  LbCas12a and 62.5 n m  crRNA (50:62.5). (b) Time courses of fluorescence detection in NEBuffer 2.1 with different concentrations of LbCas12a and crRNA with 100 n m  FQ‐labelled linker‐ssDNA. (c) Visual analysis of linker‐ssDNA degradation after 30 min of incubation with different concentrations of LbCas12a/crRNA complexes as in (b). NC, negative control. Data points represent replicates from five independent experiments, and the error bars indicate the mean ± SD. Bars carrying different letters indicate means significantly different at  P
    Nebuffer 2 1, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 32 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nebuffer 2 1/product/New England Biolabs
    Average 94 stars, based on 32 article reviews
    Price from $9.99 to $1999.99
    nebuffer 2 1 - by Bioz Stars, 2022-09
    94/100 stars

    Images

    1) Product Images from "Field detection of multiple RNA viruses/viroids in apple using a CRISPR/Cas12a‐based visual assay) Field detection of multiple RNA viruses/viroids in apple using a CRISPR/Cas12a‐based visual assay"

    Article Title: Field detection of multiple RNA viruses/viroids in apple using a CRISPR/Cas12a‐based visual assay) Field detection of multiple RNA viruses/viroids in apple using a CRISPR/Cas12a‐based visual assay

    Journal: Plant Biotechnology Journal

    doi: 10.1111/pbi.13474

    The optimization of buffer types, complex concentrations and incubation time for the trans‐cleavage activity of the LbCas12a/crRNA system. (a) Four buffers were evaluated for their effect on LbCas12a trans‐cleavage activity after 30 min of incubation with 50 n m  LbCas12a and 62.5 n m  crRNA (50:62.5). (b) Time courses of fluorescence detection in NEBuffer 2.1 with different concentrations of LbCas12a and crRNA with 100 n m  FQ‐labelled linker‐ssDNA. (c) Visual analysis of linker‐ssDNA degradation after 30 min of incubation with different concentrations of LbCas12a/crRNA complexes as in (b). NC, negative control. Data points represent replicates from five independent experiments, and the error bars indicate the mean ± SD. Bars carrying different letters indicate means significantly different at  P
    Figure Legend Snippet: The optimization of buffer types, complex concentrations and incubation time for the trans‐cleavage activity of the LbCas12a/crRNA system. (a) Four buffers were evaluated for their effect on LbCas12a trans‐cleavage activity after 30 min of incubation with 50 n m LbCas12a and 62.5 n m crRNA (50:62.5). (b) Time courses of fluorescence detection in NEBuffer 2.1 with different concentrations of LbCas12a and crRNA with 100 n m FQ‐labelled linker‐ssDNA. (c) Visual analysis of linker‐ssDNA degradation after 30 min of incubation with different concentrations of LbCas12a/crRNA complexes as in (b). NC, negative control. Data points represent replicates from five independent experiments, and the error bars indicate the mean ± SD. Bars carrying different letters indicate means significantly different at P

    Techniques Used: Incubation, Activity Assay, Fluorescence, Negative Control

    2) Product Images from "Sensitive and Specific Detection of Lumpy Skin Disease Virus in Cattle by CRISPR-Cas12a Fluorescent Assay Coupled with Recombinase Polymerase Amplification"

    Article Title: Sensitive and Specific Detection of Lumpy Skin Disease Virus in Cattle by CRISPR-Cas12a Fluorescent Assay Coupled with Recombinase Polymerase Amplification

    Journal: Genes

    doi: 10.3390/genes13050734

    Specificity of the RPA-Cas12a-fluorescence assay for detection of the LSDV. ( A ). Agarose gel electrophoresis of PCR amplification products. ( B ). Specificity of the RPA-Cas12a-fluorescence assay for LSDV detection. Lane M, DNA ladder; Lane NC, negative control; pUC57- orf068 used as a positive control; BPIV-3, Bovine parainfluenza virus type 3; BRSV, bovine respiratory syncytial virus; IBRV, Infectious Bovine Rhinotracheitis Virus; BVDV, Bovine Viral Diarrhea Virus; BCoV, Bovine coronavirus; BRV, Bovine Rotavirus; M.b, Mycoplasma bovis HB0801; M.h, A1 Mannheimia Haemolytica; STEC, Shiga toxin-producing Escherichia coli; Under blue or UV lights, the pictures were captured under blue (470 nM) and UV lights by a smartphone camera and gel imaging system.
    Figure Legend Snippet: Specificity of the RPA-Cas12a-fluorescence assay for detection of the LSDV. ( A ). Agarose gel electrophoresis of PCR amplification products. ( B ). Specificity of the RPA-Cas12a-fluorescence assay for LSDV detection. Lane M, DNA ladder; Lane NC, negative control; pUC57- orf068 used as a positive control; BPIV-3, Bovine parainfluenza virus type 3; BRSV, bovine respiratory syncytial virus; IBRV, Infectious Bovine Rhinotracheitis Virus; BVDV, Bovine Viral Diarrhea Virus; BCoV, Bovine coronavirus; BRV, Bovine Rotavirus; M.b, Mycoplasma bovis HB0801; M.h, A1 Mannheimia Haemolytica; STEC, Shiga toxin-producing Escherichia coli; Under blue or UV lights, the pictures were captured under blue (470 nM) and UV lights by a smartphone camera and gel imaging system.

    Techniques Used: Recombinase Polymerase Amplification, Fluorescence, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Amplification, Negative Control, Positive Control, Imaging

    Identification of the highly active crRNAs for LSDV orf068 gene using CRISPR-Cas12a enhanced fluorescence assay. ( A ). Schematic diagram of the position of crRNAs in LSDV orf068 gene. ( B) . Identification of the highly active crRNAs. crRNA1, 2, 3, 4, 5, 6 represent different crRNAs targeting to LSDV orf068 gene. DNA template represents PCR products of orf068 gene fragment. LSDV, lumpy skin disease virus; crRNA, CRISPR RNA; ssDNA activator is a crRNA-complementary ssDNA. Under blue or UV lights, the pictures were captured under blue (470 nM) and UV lights by a smartphone camera and gel imaging system.
    Figure Legend Snippet: Identification of the highly active crRNAs for LSDV orf068 gene using CRISPR-Cas12a enhanced fluorescence assay. ( A ). Schematic diagram of the position of crRNAs in LSDV orf068 gene. ( B) . Identification of the highly active crRNAs. crRNA1, 2, 3, 4, 5, 6 represent different crRNAs targeting to LSDV orf068 gene. DNA template represents PCR products of orf068 gene fragment. LSDV, lumpy skin disease virus; crRNA, CRISPR RNA; ssDNA activator is a crRNA-complementary ssDNA. Under blue or UV lights, the pictures were captured under blue (470 nM) and UV lights by a smartphone camera and gel imaging system.

    Techniques Used: CRISPR, Fluorescence, Polymerase Chain Reaction, Imaging

    3) Product Images from "CRISPR-Cas12a-Empowered Electrochemical Biosensor for Rapid and Ultrasensitive Detection of SARS-CoV-2 Delta Variant"

    Article Title: CRISPR-Cas12a-Empowered Electrochemical Biosensor for Rapid and Ultrasensitive Detection of SARS-CoV-2 Delta Variant

    Journal: Nano-Micro Letters

    doi: 10.1007/s40820-022-00888-4

    Feasibility and sensitivity analysis of the E-CRISPR biosensor by electrochemical method. a Electrochemical impedance spectroscopy (EIS) of AuE-AuNPs (black curve), ssDNA modified AuE-AuNPs (red curve) and ssDNA modified AuE-AuNPs after Cas12a cleavage (blue curve). b Square wave voltammetry (SWV) curves of ssDNA modified AuE-AuNPs without (black curve) and with (red curve) target. c SWV curves for a range of target DNA from 100 fM to 10 nM (red curve to purple curve), control refers to the replacement of target DNA with H 2 O. d Linear relationship between the change of current ( ΔI% ) and the logarithm of the target DNA concentration. Error bars represent standard derivations obtained in three parallel experiments
    Figure Legend Snippet: Feasibility and sensitivity analysis of the E-CRISPR biosensor by electrochemical method. a Electrochemical impedance spectroscopy (EIS) of AuE-AuNPs (black curve), ssDNA modified AuE-AuNPs (red curve) and ssDNA modified AuE-AuNPs after Cas12a cleavage (blue curve). b Square wave voltammetry (SWV) curves of ssDNA modified AuE-AuNPs without (black curve) and with (red curve) target. c SWV curves for a range of target DNA from 100 fM to 10 nM (red curve to purple curve), control refers to the replacement of target DNA with H 2 O. d Linear relationship between the change of current ( ΔI% ) and the logarithm of the target DNA concentration. Error bars represent standard derivations obtained in three parallel experiments

    Techniques Used: CRISPR, Impedance Spectroscopy, Modification, Concentration Assay

    4) Product Images from "Isothermal Amplification and Ambient Visualization in a Single Tube for the Detection of SARS-CoV-2 Using Loop-Mediated Amplification and CRISPR Technology"

    Article Title: Isothermal Amplification and Ambient Visualization in a Single Tube for the Detection of SARS-CoV-2 Using Loop-Mediated Amplification and CRISPR Technology

    Journal: Analytical Chemistry

    doi: 10.1021/acs.analchem.0c04047

    Incorporating CRISPR Cas12a-mediated detection with RT-LAMP amplification improves the specificity. (A) RT-LAMP amplification curves from triplicate analyses of the target N gene and the negative controls, with the real-time fluorescence detection of intercalating SYBR Green. (B) CRISPR Cas12a-mediated detection of the RT-LAMP products. In this set of experiments, 25 μL of RT-LAMP reaction solution contained 1× NEBuffer 2.1 buffer, 1.4 mM deoxynucleotide (dNTP), 0.2 μM each of the outer primers (F3 and B3), 1.6 μM each of the inner primers (FIP and BIP), 0.8 μM each of the loop primers (LF and LB), 4 units of RNase inhibitor, 7.5 units of WarmStart RTx reverse transcriptase, 8 units of Bst 2.0 DNA polymerase, and 5 μL of 750 copies/μL viral RNA (as target) or nuclease-free water (as negative control). RT-LAMP reactions were performed at 62 °C, and the reaction products were monitored using either SYBR Green (A) or the CRISPR Cas12a system (B).
    Figure Legend Snippet: Incorporating CRISPR Cas12a-mediated detection with RT-LAMP amplification improves the specificity. (A) RT-LAMP amplification curves from triplicate analyses of the target N gene and the negative controls, with the real-time fluorescence detection of intercalating SYBR Green. (B) CRISPR Cas12a-mediated detection of the RT-LAMP products. In this set of experiments, 25 μL of RT-LAMP reaction solution contained 1× NEBuffer 2.1 buffer, 1.4 mM deoxynucleotide (dNTP), 0.2 μM each of the outer primers (F3 and B3), 1.6 μM each of the inner primers (FIP and BIP), 0.8 μM each of the loop primers (LF and LB), 4 units of RNase inhibitor, 7.5 units of WarmStart RTx reverse transcriptase, 8 units of Bst 2.0 DNA polymerase, and 5 μL of 750 copies/μL viral RNA (as target) or nuclease-free water (as negative control). RT-LAMP reactions were performed at 62 °C, and the reaction products were monitored using either SYBR Green (A) or the CRISPR Cas12a system (B).

    Techniques Used: CRISPR, Amplification, Fluorescence, SYBR Green Assay, Negative Control

    5) Product Images from "Field detection of multiple RNA viruses/viroids in apple using a CRISPR/Cas12a‐based visual assay) Field detection of multiple RNA viruses/viroids in apple using a CRISPR/Cas12a‐based visual assay"

    Article Title: Field detection of multiple RNA viruses/viroids in apple using a CRISPR/Cas12a‐based visual assay) Field detection of multiple RNA viruses/viroids in apple using a CRISPR/Cas12a‐based visual assay

    Journal: Plant Biotechnology Journal

    doi: 10.1111/pbi.13474

    The optimization of buffer types, complex concentrations and incubation time for the trans‐cleavage activity of the LbCas12a/crRNA system. (a) Four buffers were evaluated for their effect on LbCas12a trans‐cleavage activity after 30 min of incubation with 50 n m  LbCas12a and 62.5 n m  crRNA (50:62.5). (b) Time courses of fluorescence detection in NEBuffer 2.1 with different concentrations of LbCas12a and crRNA with 100 n m  FQ‐labelled linker‐ssDNA. (c) Visual analysis of linker‐ssDNA degradation after 30 min of incubation with different concentrations of LbCas12a/crRNA complexes as in (b). NC, negative control. Data points represent replicates from five independent experiments, and the error bars indicate the mean ± SD. Bars carrying different letters indicate means significantly different at  P
    Figure Legend Snippet: The optimization of buffer types, complex concentrations and incubation time for the trans‐cleavage activity of the LbCas12a/crRNA system. (a) Four buffers were evaluated for their effect on LbCas12a trans‐cleavage activity after 30 min of incubation with 50 n m LbCas12a and 62.5 n m crRNA (50:62.5). (b) Time courses of fluorescence detection in NEBuffer 2.1 with different concentrations of LbCas12a and crRNA with 100 n m FQ‐labelled linker‐ssDNA. (c) Visual analysis of linker‐ssDNA degradation after 30 min of incubation with different concentrations of LbCas12a/crRNA complexes as in (b). NC, negative control. Data points represent replicates from five independent experiments, and the error bars indicate the mean ± SD. Bars carrying different letters indicate means significantly different at P

    Techniques Used: Incubation, Activity Assay, Fluorescence, Negative Control

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    New England Biolabs nebuffer 2 1
    The optimization of buffer types, complex concentrations and incubation time for the trans‐cleavage activity of the LbCas12a/crRNA system. (a) Four buffers were evaluated for their effect on LbCas12a trans‐cleavage activity after 30 min of incubation with 50 n m  LbCas12a and 62.5 n m  crRNA (50:62.5). (b) Time courses of fluorescence detection in NEBuffer 2.1 with different concentrations of LbCas12a and crRNA with 100 n m  FQ‐labelled linker‐ssDNA. (c) Visual analysis of linker‐ssDNA degradation after 30 min of incubation with different concentrations of LbCas12a/crRNA complexes as in (b). NC, negative control. Data points represent replicates from five independent experiments, and the error bars indicate the mean ± SD. Bars carrying different letters indicate means significantly different at  P
    Nebuffer 2 1, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nebuffer 2 1/product/New England Biolabs
    Average 97 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    nebuffer 2 1 - by Bioz Stars, 2022-09
    97/100 stars
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    The optimization of buffer types, complex concentrations and incubation time for the trans‐cleavage activity of the LbCas12a/crRNA system. (a) Four buffers were evaluated for their effect on LbCas12a trans‐cleavage activity after 30 min of incubation with 50 n m  LbCas12a and 62.5 n m  crRNA (50:62.5). (b) Time courses of fluorescence detection in NEBuffer 2.1 with different concentrations of LbCas12a and crRNA with 100 n m  FQ‐labelled linker‐ssDNA. (c) Visual analysis of linker‐ssDNA degradation after 30 min of incubation with different concentrations of LbCas12a/crRNA complexes as in (b). NC, negative control. Data points represent replicates from five independent experiments, and the error bars indicate the mean ± SD. Bars carrying different letters indicate means significantly different at  P

    Journal: Plant Biotechnology Journal

    Article Title: Field detection of multiple RNA viruses/viroids in apple using a CRISPR/Cas12a‐based visual assay) Field detection of multiple RNA viruses/viroids in apple using a CRISPR/Cas12a‐based visual assay

    doi: 10.1111/pbi.13474

    Figure Lengend Snippet: The optimization of buffer types, complex concentrations and incubation time for the trans‐cleavage activity of the LbCas12a/crRNA system. (a) Four buffers were evaluated for their effect on LbCas12a trans‐cleavage activity after 30 min of incubation with 50 n m LbCas12a and 62.5 n m crRNA (50:62.5). (b) Time courses of fluorescence detection in NEBuffer 2.1 with different concentrations of LbCas12a and crRNA with 100 n m FQ‐labelled linker‐ssDNA. (c) Visual analysis of linker‐ssDNA degradation after 30 min of incubation with different concentrations of LbCas12a/crRNA complexes as in (b). NC, negative control. Data points represent replicates from five independent experiments, and the error bars indicate the mean ± SD. Bars carrying different letters indicate means significantly different at P

    Article Snippet: Unless otherwise described, the integrated LbCas12a/crRNA fluorescence and visualization detection procedures were as follows: the optimized assays were performed with 50 nm LbCas12a, 62.5 nm crRNA, 100 nm of (FQ)‐labelled linker‐ssDNA, 2.5 μL of nucleic acid target and add NEBuffer 2.1 up to 25 μL.

    Techniques: Incubation, Activity Assay, Fluorescence, Negative Control

    Specificity of the RPA-Cas12a-fluorescence assay for detection of the LSDV. ( A ). Agarose gel electrophoresis of PCR amplification products. ( B ). Specificity of the RPA-Cas12a-fluorescence assay for LSDV detection. Lane M, DNA ladder; Lane NC, negative control; pUC57- orf068 used as a positive control; BPIV-3, Bovine parainfluenza virus type 3; BRSV, bovine respiratory syncytial virus; IBRV, Infectious Bovine Rhinotracheitis Virus; BVDV, Bovine Viral Diarrhea Virus; BCoV, Bovine coronavirus; BRV, Bovine Rotavirus; M.b, Mycoplasma bovis HB0801; M.h, A1 Mannheimia Haemolytica; STEC, Shiga toxin-producing Escherichia coli; Under blue or UV lights, the pictures were captured under blue (470 nM) and UV lights by a smartphone camera and gel imaging system.

    Journal: Genes

    Article Title: Sensitive and Specific Detection of Lumpy Skin Disease Virus in Cattle by CRISPR-Cas12a Fluorescent Assay Coupled with Recombinase Polymerase Amplification

    doi: 10.3390/genes13050734

    Figure Lengend Snippet: Specificity of the RPA-Cas12a-fluorescence assay for detection of the LSDV. ( A ). Agarose gel electrophoresis of PCR amplification products. ( B ). Specificity of the RPA-Cas12a-fluorescence assay for LSDV detection. Lane M, DNA ladder; Lane NC, negative control; pUC57- orf068 used as a positive control; BPIV-3, Bovine parainfluenza virus type 3; BRSV, bovine respiratory syncytial virus; IBRV, Infectious Bovine Rhinotracheitis Virus; BVDV, Bovine Viral Diarrhea Virus; BCoV, Bovine coronavirus; BRV, Bovine Rotavirus; M.b, Mycoplasma bovis HB0801; M.h, A1 Mannheimia Haemolytica; STEC, Shiga toxin-producing Escherichia coli; Under blue or UV lights, the pictures were captured under blue (470 nM) and UV lights by a smartphone camera and gel imaging system.

    Article Snippet: 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.

    Techniques: Recombinase Polymerase Amplification, Fluorescence, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Amplification, Negative Control, Positive Control, Imaging

    Identification of the highly active crRNAs for LSDV orf068 gene using CRISPR-Cas12a enhanced fluorescence assay. ( A ). Schematic diagram of the position of crRNAs in LSDV orf068 gene. ( B) . Identification of the highly active crRNAs. crRNA1, 2, 3, 4, 5, 6 represent different crRNAs targeting to LSDV orf068 gene. DNA template represents PCR products of orf068 gene fragment. LSDV, lumpy skin disease virus; crRNA, CRISPR RNA; ssDNA activator is a crRNA-complementary ssDNA. Under blue or UV lights, the pictures were captured under blue (470 nM) and UV lights by a smartphone camera and gel imaging system.

    Journal: Genes

    Article Title: Sensitive and Specific Detection of Lumpy Skin Disease Virus in Cattle by CRISPR-Cas12a Fluorescent Assay Coupled with Recombinase Polymerase Amplification

    doi: 10.3390/genes13050734

    Figure Lengend Snippet: Identification of the highly active crRNAs for LSDV orf068 gene using CRISPR-Cas12a enhanced fluorescence assay. ( A ). Schematic diagram of the position of crRNAs in LSDV orf068 gene. ( B) . Identification of the highly active crRNAs. crRNA1, 2, 3, 4, 5, 6 represent different crRNAs targeting to LSDV orf068 gene. DNA template represents PCR products of orf068 gene fragment. LSDV, lumpy skin disease virus; crRNA, CRISPR RNA; ssDNA activator is a crRNA-complementary ssDNA. Under blue or UV lights, the pictures were captured under blue (470 nM) and UV lights by a smartphone camera and gel imaging system.

    Article Snippet: 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.

    Techniques: CRISPR, Fluorescence, Polymerase Chain Reaction, Imaging

    Feasibility and sensitivity analysis of the E-CRISPR biosensor by electrochemical method. a Electrochemical impedance spectroscopy (EIS) of AuE-AuNPs (black curve), ssDNA modified AuE-AuNPs (red curve) and ssDNA modified AuE-AuNPs after Cas12a cleavage (blue curve). b Square wave voltammetry (SWV) curves of ssDNA modified AuE-AuNPs without (black curve) and with (red curve) target. c SWV curves for a range of target DNA from 100 fM to 10 nM (red curve to purple curve), control refers to the replacement of target DNA with H 2 O. d Linear relationship between the change of current ( ΔI% ) and the logarithm of the target DNA concentration. Error bars represent standard derivations obtained in three parallel experiments

    Journal: Nano-Micro Letters

    Article Title: CRISPR-Cas12a-Empowered Electrochemical Biosensor for Rapid and Ultrasensitive Detection of SARS-CoV-2 Delta Variant

    doi: 10.1007/s40820-022-00888-4

    Figure Lengend Snippet: Feasibility and sensitivity analysis of the E-CRISPR biosensor by electrochemical method. a Electrochemical impedance spectroscopy (EIS) of AuE-AuNPs (black curve), ssDNA modified AuE-AuNPs (red curve) and ssDNA modified AuE-AuNPs after Cas12a cleavage (blue curve). b Square wave voltammetry (SWV) curves of ssDNA modified AuE-AuNPs without (black curve) and with (red curve) target. c SWV curves for a range of target DNA from 100 fM to 10 nM (red curve to purple curve), control refers to the replacement of target DNA with H 2 O. d Linear relationship between the change of current ( ΔI% ) and the logarithm of the target DNA concentration. Error bars represent standard derivations obtained in three parallel experiments

    Article Snippet: For electrochemical analysis of trans-cleaved reporters, the Cas12a-mediated cleavage assay was carried out by adding a Cas12a-crRNA duplex (100 nM Cas12a, 100 nM crRNA, 1× NEBuffer 2.1) into target DNA to form Cas12a-crRNA-target DNA triplex.

    Techniques: CRISPR, Impedance Spectroscopy, Modification, Concentration Assay

    Incorporating CRISPR Cas12a-mediated detection with RT-LAMP amplification improves the specificity. (A) RT-LAMP amplification curves from triplicate analyses of the target N gene and the negative controls, with the real-time fluorescence detection of intercalating SYBR Green. (B) CRISPR Cas12a-mediated detection of the RT-LAMP products. In this set of experiments, 25 μL of RT-LAMP reaction solution contained 1× NEBuffer 2.1 buffer, 1.4 mM deoxynucleotide (dNTP), 0.2 μM each of the outer primers (F3 and B3), 1.6 μM each of the inner primers (FIP and BIP), 0.8 μM each of the loop primers (LF and LB), 4 units of RNase inhibitor, 7.5 units of WarmStart RTx reverse transcriptase, 8 units of Bst 2.0 DNA polymerase, and 5 μL of 750 copies/μL viral RNA (as target) or nuclease-free water (as negative control). RT-LAMP reactions were performed at 62 °C, and the reaction products were monitored using either SYBR Green (A) or the CRISPR Cas12a system (B).

    Journal: Analytical Chemistry

    Article Title: Isothermal Amplification and Ambient Visualization in a Single Tube for the Detection of SARS-CoV-2 Using Loop-Mediated Amplification and CRISPR Technology

    doi: 10.1021/acs.analchem.0c04047

    Figure Lengend Snippet: Incorporating CRISPR Cas12a-mediated detection with RT-LAMP amplification improves the specificity. (A) RT-LAMP amplification curves from triplicate analyses of the target N gene and the negative controls, with the real-time fluorescence detection of intercalating SYBR Green. (B) CRISPR Cas12a-mediated detection of the RT-LAMP products. In this set of experiments, 25 μL of RT-LAMP reaction solution contained 1× NEBuffer 2.1 buffer, 1.4 mM deoxynucleotide (dNTP), 0.2 μM each of the outer primers (F3 and B3), 1.6 μM each of the inner primers (FIP and BIP), 0.8 μM each of the loop primers (LF and LB), 4 units of RNase inhibitor, 7.5 units of WarmStart RTx reverse transcriptase, 8 units of Bst 2.0 DNA polymerase, and 5 μL of 750 copies/μL viral RNA (as target) or nuclease-free water (as negative control). RT-LAMP reactions were performed at 62 °C, and the reaction products were monitored using either SYBR Green (A) or the CRISPR Cas12a system (B).

    Article Snippet: The EnGen Lba Cas12a enzyme (NEB) at 1 μM concentration was preincubated with 1.25 μM gRNA in 1× NEBuffer 2.1 (NEB) to form the ribonucleoprotein (RNP) complex.

    Techniques: CRISPR, Amplification, Fluorescence, SYBR Green Assay, Negative Control