1x nebuffer 2 1  (New England Biolabs)


Bioz Verified Symbol New England Biolabs is a verified supplier
Bioz Manufacturer Symbol New England Biolabs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 97

    Structured Review

    New England Biolabs 1x nebuffer 2 1
    BrCas12b characterization and one-pot specificity    sensitivity testing. ( a ) Schematic of binary complex illustrating cleavage pattern of dsDNA target. ( b ) Temperature-dependent in-vitro cleavage assay of AacCas12b, AapCas12b, and BrCas12b targeting more restrictive PAM TTTG. 125 nM sgRNA:100 nM Cas12b:7 nM dsDNA was combined in 1X NEBuffer 2.1. The experiment was repeated (n = 2) with similar results. ( c ) Michaelis-Menten Kinetics of BrCas12b trans-cleavage at 62°C. ( d ) Differential scanning fluorimetry of AacCas12b, AapCas12b, and BrCas12b complexes.  (e)  Multiplexing using FITC to detect LAMP amplification and HEX to monitor trans-cleavage of BrCas12b. ( f ) – ( i ) Detection capability of BrCas12b via trans-cleavage against AacCas12b and AapCas12b at various temperatures. Fluorescence kinetics within 30 minutes was monitored using a HEX-based reporter. Shaded regions represent standard deviation (n = 3 replicates). STOPCovid LAMP primers were used in (f) to compare performance with AapCas12b. A different set of LAMP primers (DETECTR) with optimal performance at a temperature higher than 60°C were used in (g), (h), and (i). (+) denotes the presence of the SARS-CoV-2 genomic RNA control, and (-) signifies the non-template control (NTC). ( j ) – ( m ) Specificity testing using a pair of sgRNA and LAMP primers targeting SARS-CoV-2 variants (n = 3 replicates). ( n ) – ( q ) Limit of detection on N gene and S gene targeting alpha (B.1.1.7), beta (B.1.352), and delta (B.1.617.2), respectively (n = 3 replicates).
    1x Nebuffer 2 1, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/1x nebuffer 2 1/product/New England Biolabs
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    1x nebuffer 2 1 - by Bioz Stars, 2022-07
    97/100 stars

    Images

    1) Product Images from "A Thermostable Cas12b from Brevibacillus Leverages One-pot Detection of SARS-CoV-2 Variants of Concern"

    Article Title: A Thermostable Cas12b from Brevibacillus Leverages One-pot Detection of SARS-CoV-2 Variants of Concern

    Journal: medRxiv

    doi: 10.1101/2021.10.15.21265066

    BrCas12b characterization and one-pot specificity    sensitivity testing. ( a ) Schematic of binary complex illustrating cleavage pattern of dsDNA target. ( b ) Temperature-dependent in-vitro cleavage assay of AacCas12b, AapCas12b, and BrCas12b targeting more restrictive PAM TTTG. 125 nM sgRNA:100 nM Cas12b:7 nM dsDNA was combined in 1X NEBuffer 2.1. The experiment was repeated (n = 2) with similar results. ( c ) Michaelis-Menten Kinetics of BrCas12b trans-cleavage at 62°C. ( d ) Differential scanning fluorimetry of AacCas12b, AapCas12b, and BrCas12b complexes.  (e)  Multiplexing using FITC to detect LAMP amplification and HEX to monitor trans-cleavage of BrCas12b. ( f ) – ( i ) Detection capability of BrCas12b via trans-cleavage against AacCas12b and AapCas12b at various temperatures. Fluorescence kinetics within 30 minutes was monitored using a HEX-based reporter. Shaded regions represent standard deviation (n = 3 replicates). STOPCovid LAMP primers were used in (f) to compare performance with AapCas12b. A different set of LAMP primers (DETECTR) with optimal performance at a temperature higher than 60°C were used in (g), (h), and (i). (+) denotes the presence of the SARS-CoV-2 genomic RNA control, and (-) signifies the non-template control (NTC). ( j ) – ( m ) Specificity testing using a pair of sgRNA and LAMP primers targeting SARS-CoV-2 variants (n = 3 replicates). ( n ) – ( q ) Limit of detection on N gene and S gene targeting alpha (B.1.1.7), beta (B.1.352), and delta (B.1.617.2), respectively (n = 3 replicates).
    Figure Legend Snippet: BrCas12b characterization and one-pot specificity sensitivity testing. ( a ) Schematic of binary complex illustrating cleavage pattern of dsDNA target. ( b ) Temperature-dependent in-vitro cleavage assay of AacCas12b, AapCas12b, and BrCas12b targeting more restrictive PAM TTTG. 125 nM sgRNA:100 nM Cas12b:7 nM dsDNA was combined in 1X NEBuffer 2.1. The experiment was repeated (n = 2) with similar results. ( c ) Michaelis-Menten Kinetics of BrCas12b trans-cleavage at 62°C. ( d ) Differential scanning fluorimetry of AacCas12b, AapCas12b, and BrCas12b complexes. (e) Multiplexing using FITC to detect LAMP amplification and HEX to monitor trans-cleavage of BrCas12b. ( f ) – ( i ) Detection capability of BrCas12b via trans-cleavage against AacCas12b and AapCas12b at various temperatures. Fluorescence kinetics within 30 minutes was monitored using a HEX-based reporter. Shaded regions represent standard deviation (n = 3 replicates). STOPCovid LAMP primers were used in (f) to compare performance with AapCas12b. A different set of LAMP primers (DETECTR) with optimal performance at a temperature higher than 60°C were used in (g), (h), and (i). (+) denotes the presence of the SARS-CoV-2 genomic RNA control, and (-) signifies the non-template control (NTC). ( j ) – ( m ) Specificity testing using a pair of sgRNA and LAMP primers targeting SARS-CoV-2 variants (n = 3 replicates). ( n ) – ( q ) Limit of detection on N gene and S gene targeting alpha (B.1.1.7), beta (B.1.352), and delta (B.1.617.2), respectively (n = 3 replicates).

    Techniques Used: In Vitro, Cleavage Assay, Multiplexing, Amplification, Fluorescence, Standard Deviation

    2) Product Images from "A thermostable Cas12b from Brevibacillus leverages one-pot discrimination of SARS-CoV-2 variants of concern"

    Article Title: A thermostable Cas12b from Brevibacillus leverages one-pot discrimination of SARS-CoV-2 variants of concern

    Journal: EBioMedicine

    doi: 10.1016/j.ebiom.2022.103926

    BrCas12b characterisation and one-pot specificity    sensitivity testing. (a) Schematic of binary complex illustrating cleavage pattern of dsDNA target. (b) Temperature-dependent  in-vitro  cleavage assay of AacCas12b, AapCas12b, and BrCas12b targeting more restrictive PAM TTTG. 125 nM sgRNA:100 nM Cas12b:7 nM dsDNA was combined in 1X NEBuffer 2·1. The experiment was repeated ( n  = 2 biological replicates) with similar results. (c) Michaelis-Menten Kinetics of BrCas12b trans-cleavage at 62 °C. (d) Differential scanning fluorimetry of apo AacCas12b, apo AapCas12b, and apo BrCas12b and their corresponding Cas12b:sgRNA complexes. (e) Multiplexing using FITC to detect LAMP amplification and HEX to monitor trans-cleavage of BrCas12b. (f)–(i) Detection capability of BrCas12b via trans-cleavage against AacCas12b and AapCas12b at various temperatures. Fluorescence kinetics within 30 min was monitored using HEX-based reporter. Shaded regions represent standard deviation ( n  = 3 biological replicates). STOPCovid LAMP primers were used in (f) to compare performance with AapCas12b. A different set of LAMP primers (DETECTR) with optimal performance at temperature higher than 60 °C were used in (g), (h), and (i). (+) denotes the presence of the SARS-CoV-2 genomic RNA control, and (-) signifies the non-template control (NTC). (j)–(n) Specificity testing using a pair of sgRNA and LAMP primers targeting SARS-CoV-2 variants of concern ( n  = 3 biological replicates). (o) – (t) Limit of detection of non-N gene targeting Alpha (B.1.1.7), Beta (B.1.352), Gamma (P1), Delta (B.1.617.2), Omicron (B.1.1.529) and N gene, respectively ( n  = 3 biological replicates).
    Figure Legend Snippet: BrCas12b characterisation and one-pot specificity sensitivity testing. (a) Schematic of binary complex illustrating cleavage pattern of dsDNA target. (b) Temperature-dependent in-vitro cleavage assay of AacCas12b, AapCas12b, and BrCas12b targeting more restrictive PAM TTTG. 125 nM sgRNA:100 nM Cas12b:7 nM dsDNA was combined in 1X NEBuffer 2·1. The experiment was repeated ( n  = 2 biological replicates) with similar results. (c) Michaelis-Menten Kinetics of BrCas12b trans-cleavage at 62 °C. (d) Differential scanning fluorimetry of apo AacCas12b, apo AapCas12b, and apo BrCas12b and their corresponding Cas12b:sgRNA complexes. (e) Multiplexing using FITC to detect LAMP amplification and HEX to monitor trans-cleavage of BrCas12b. (f)–(i) Detection capability of BrCas12b via trans-cleavage against AacCas12b and AapCas12b at various temperatures. Fluorescence kinetics within 30 min was monitored using HEX-based reporter. Shaded regions represent standard deviation ( n  = 3 biological replicates). STOPCovid LAMP primers were used in (f) to compare performance with AapCas12b. A different set of LAMP primers (DETECTR) with optimal performance at temperature higher than 60 °C were used in (g), (h), and (i). (+) denotes the presence of the SARS-CoV-2 genomic RNA control, and (-) signifies the non-template control (NTC). (j)–(n) Specificity testing using a pair of sgRNA and LAMP primers targeting SARS-CoV-2 variants of concern ( n  = 3 biological replicates). (o) – (t) Limit of detection of non-N gene targeting Alpha (B.1.1.7), Beta (B.1.352), Gamma (P1), Delta (B.1.617.2), Omicron (B.1.1.529) and N gene, respectively ( n  = 3 biological replicates).

    Techniques Used: In Vitro, Cleavage Assay, Multiplexing, Amplification, Fluorescence, Standard Deviation

    3) Product Images from "A thermostable Cas12b from Brevibacillus leverages one-pot discrimination of SARS-CoV-2 variants of concern"

    Article Title: A thermostable Cas12b from Brevibacillus leverages one-pot discrimination of SARS-CoV-2 variants of concern

    Journal: EBioMedicine

    doi: 10.1016/j.ebiom.2022.103926

    BrCas12b characterisation and one-pot specificity    sensitivity testing. (a) Schematic of binary complex illustrating cleavage pattern of dsDNA target. (b) Temperature-dependent  in-vitro  cleavage assay of AacCas12b, AapCas12b, and BrCas12b targeting more restrictive PAM TTTG. 125 nM sgRNA:100 nM Cas12b:7 nM dsDNA was combined in 1X NEBuffer 2·1. The experiment was repeated ( n  = 2 biological replicates) with similar results. (c) Michaelis-Menten Kinetics of BrCas12b trans-cleavage at 62 °C. (d) Differential scanning fluorimetry of apo AacCas12b, apo AapCas12b, and apo BrCas12b and their corresponding Cas12b:sgRNA complexes. (e) Multiplexing using FITC to detect LAMP amplification and HEX to monitor trans-cleavage of BrCas12b. (f)–(i) Detection capability of BrCas12b via trans-cleavage against AacCas12b and AapCas12b at various temperatures. Fluorescence kinetics within 30 min was monitored using HEX-based reporter. Shaded regions represent standard deviation ( n  = 3 biological replicates). STOPCovid LAMP primers were used in (f) to compare performance with AapCas12b. A different set of LAMP primers (DETECTR) with optimal performance at temperature higher than 60 °C were used in (g), (h), and (i). (+) denotes the presence of the SARS-CoV-2 genomic RNA control, and (-) signifies the non-template control (NTC). (j)–(n) Specificity testing using a pair of sgRNA and LAMP primers targeting SARS-CoV-2 variants of concern ( n  = 3 biological replicates). (o) – (t) Limit of detection of non-N gene targeting Alpha (B.1.1.7), Beta (B.1.352), Gamma (P1), Delta (B.1.617.2), Omicron (B.1.1.529) and N gene, respectively ( n  = 3 biological replicates).
    Figure Legend Snippet: BrCas12b characterisation and one-pot specificity sensitivity testing. (a) Schematic of binary complex illustrating cleavage pattern of dsDNA target. (b) Temperature-dependent in-vitro cleavage assay of AacCas12b, AapCas12b, and BrCas12b targeting more restrictive PAM TTTG. 125 nM sgRNA:100 nM Cas12b:7 nM dsDNA was combined in 1X NEBuffer 2·1. The experiment was repeated ( n  = 2 biological replicates) with similar results. (c) Michaelis-Menten Kinetics of BrCas12b trans-cleavage at 62 °C. (d) Differential scanning fluorimetry of apo AacCas12b, apo AapCas12b, and apo BrCas12b and their corresponding Cas12b:sgRNA complexes. (e) Multiplexing using FITC to detect LAMP amplification and HEX to monitor trans-cleavage of BrCas12b. (f)–(i) Detection capability of BrCas12b via trans-cleavage against AacCas12b and AapCas12b at various temperatures. Fluorescence kinetics within 30 min was monitored using HEX-based reporter. Shaded regions represent standard deviation ( n  = 3 biological replicates). STOPCovid LAMP primers were used in (f) to compare performance with AapCas12b. A different set of LAMP primers (DETECTR) with optimal performance at temperature higher than 60 °C were used in (g), (h), and (i). (+) denotes the presence of the SARS-CoV-2 genomic RNA control, and (-) signifies the non-template control (NTC). (j)–(n) Specificity testing using a pair of sgRNA and LAMP primers targeting SARS-CoV-2 variants of concern ( n  = 3 biological replicates). (o) – (t) Limit of detection of non-N gene targeting Alpha (B.1.1.7), Beta (B.1.352), Gamma (P1), Delta (B.1.617.2), Omicron (B.1.1.529) and N gene, respectively ( n  = 3 biological replicates).

    Techniques Used: In Vitro, Cleavage Assay, Multiplexing, Amplification, Fluorescence, Standard Deviation

    4) Product Images from "A Thermostable Cas12b from Brevibacillus Leverages One-pot Detection of SARS-CoV-2 Variants of Concern"

    Article Title: A Thermostable Cas12b from Brevibacillus Leverages One-pot Detection of SARS-CoV-2 Variants of Concern

    Journal: medRxiv

    doi: 10.1101/2021.10.15.21265066

    BrCas12b characterization and one-pot specificity    sensitivity testing. ( a ) Schematic of binary complex illustrating cleavage pattern of dsDNA target. ( b ) Temperature-dependent in-vitro cleavage assay of AacCas12b, AapCas12b, and BrCas12b targeting more restrictive PAM TTTG. 125 nM sgRNA:100 nM Cas12b:7 nM dsDNA was combined in 1X NEBuffer 2.1. The experiment was repeated (n = 2) with similar results. ( c ) Michaelis-Menten Kinetics of BrCas12b trans-cleavage at 62°C. ( d ) Differential scanning fluorimetry of AacCas12b, AapCas12b, and BrCas12b complexes.  (e)  Multiplexing using FITC to detect LAMP amplification and HEX to monitor trans-cleavage of BrCas12b. ( f ) – ( i ) Detection capability of BrCas12b via trans-cleavage against AacCas12b and AapCas12b at various temperatures. Fluorescence kinetics within 30 minutes was monitored using a HEX-based reporter. Shaded regions represent standard deviation (n = 3 replicates). STOPCovid LAMP primers were used in (f) to compare performance with AapCas12b. A different set of LAMP primers (DETECTR) with optimal performance at a temperature higher than 60°C were used in (g), (h), and (i). (+) denotes the presence of the SARS-CoV-2 genomic RNA control, and (-) signifies the non-template control (NTC). ( j ) – ( m ) Specificity testing using a pair of sgRNA and LAMP primers targeting SARS-CoV-2 variants (n = 3 replicates). ( n ) – ( q ) Limit of detection on N gene and S gene targeting alpha (B.1.1.7), beta (B.1.352), and delta (B.1.617.2), respectively (n = 3 replicates).
    Figure Legend Snippet: BrCas12b characterization and one-pot specificity sensitivity testing. ( a ) Schematic of binary complex illustrating cleavage pattern of dsDNA target. ( b ) Temperature-dependent in-vitro cleavage assay of AacCas12b, AapCas12b, and BrCas12b targeting more restrictive PAM TTTG. 125 nM sgRNA:100 nM Cas12b:7 nM dsDNA was combined in 1X NEBuffer 2.1. The experiment was repeated (n = 2) with similar results. ( c ) Michaelis-Menten Kinetics of BrCas12b trans-cleavage at 62°C. ( d ) Differential scanning fluorimetry of AacCas12b, AapCas12b, and BrCas12b complexes. (e) Multiplexing using FITC to detect LAMP amplification and HEX to monitor trans-cleavage of BrCas12b. ( f ) – ( i ) Detection capability of BrCas12b via trans-cleavage against AacCas12b and AapCas12b at various temperatures. Fluorescence kinetics within 30 minutes was monitored using a HEX-based reporter. Shaded regions represent standard deviation (n = 3 replicates). STOPCovid LAMP primers were used in (f) to compare performance with AapCas12b. A different set of LAMP primers (DETECTR) with optimal performance at a temperature higher than 60°C were used in (g), (h), and (i). (+) denotes the presence of the SARS-CoV-2 genomic RNA control, and (-) signifies the non-template control (NTC). ( j ) – ( m ) Specificity testing using a pair of sgRNA and LAMP primers targeting SARS-CoV-2 variants (n = 3 replicates). ( n ) – ( q ) Limit of detection on N gene and S gene targeting alpha (B.1.1.7), beta (B.1.352), and delta (B.1.617.2), respectively (n = 3 replicates).

    Techniques Used: In Vitro, Cleavage Assay, Multiplexing, Amplification, Fluorescence, Standard Deviation

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 97
    New England Biolabs 1x nebuffer 2 1
    BrCas12b characterization and one-pot specificity    sensitivity testing. ( a ) Schematic of binary complex illustrating cleavage pattern of dsDNA target. ( b ) Temperature-dependent in-vitro cleavage assay of AacCas12b, AapCas12b, and BrCas12b targeting more restrictive PAM TTTG. 125 nM sgRNA:100 nM Cas12b:7 nM dsDNA was combined in 1X NEBuffer 2.1. The experiment was repeated (n = 2) with similar results. ( c ) Michaelis-Menten Kinetics of BrCas12b trans-cleavage at 62°C. ( d ) Differential scanning fluorimetry of AacCas12b, AapCas12b, and BrCas12b complexes.  (e)  Multiplexing using FITC to detect LAMP amplification and HEX to monitor trans-cleavage of BrCas12b. ( f ) – ( i ) Detection capability of BrCas12b via trans-cleavage against AacCas12b and AapCas12b at various temperatures. Fluorescence kinetics within 30 minutes was monitored using a HEX-based reporter. Shaded regions represent standard deviation (n = 3 replicates). STOPCovid LAMP primers were used in (f) to compare performance with AapCas12b. A different set of LAMP primers (DETECTR) with optimal performance at a temperature higher than 60°C were used in (g), (h), and (i). (+) denotes the presence of the SARS-CoV-2 genomic RNA control, and (-) signifies the non-template control (NTC). ( j ) – ( m ) Specificity testing using a pair of sgRNA and LAMP primers targeting SARS-CoV-2 variants (n = 3 replicates). ( n ) – ( q ) Limit of detection on N gene and S gene targeting alpha (B.1.1.7), beta (B.1.352), and delta (B.1.617.2), respectively (n = 3 replicates).
    1x Nebuffer 2 1, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/1x nebuffer 2 1/product/New England Biolabs
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    1x nebuffer 2 1 - by Bioz Stars, 2022-07
    97/100 stars
      Buy from Supplier

    Image Search Results


    BrCas12b characterization and one-pot specificity    sensitivity testing. ( a ) Schematic of binary complex illustrating cleavage pattern of dsDNA target. ( b ) Temperature-dependent in-vitro cleavage assay of AacCas12b, AapCas12b, and BrCas12b targeting more restrictive PAM TTTG. 125 nM sgRNA:100 nM Cas12b:7 nM dsDNA was combined in 1X NEBuffer 2.1. The experiment was repeated (n = 2) with similar results. ( c ) Michaelis-Menten Kinetics of BrCas12b trans-cleavage at 62°C. ( d ) Differential scanning fluorimetry of AacCas12b, AapCas12b, and BrCas12b complexes.  (e)  Multiplexing using FITC to detect LAMP amplification and HEX to monitor trans-cleavage of BrCas12b. ( f ) – ( i ) Detection capability of BrCas12b via trans-cleavage against AacCas12b and AapCas12b at various temperatures. Fluorescence kinetics within 30 minutes was monitored using a HEX-based reporter. Shaded regions represent standard deviation (n = 3 replicates). STOPCovid LAMP primers were used in (f) to compare performance with AapCas12b. A different set of LAMP primers (DETECTR) with optimal performance at a temperature higher than 60°C were used in (g), (h), and (i). (+) denotes the presence of the SARS-CoV-2 genomic RNA control, and (-) signifies the non-template control (NTC). ( j ) – ( m ) Specificity testing using a pair of sgRNA and LAMP primers targeting SARS-CoV-2 variants (n = 3 replicates). ( n ) – ( q ) Limit of detection on N gene and S gene targeting alpha (B.1.1.7), beta (B.1.352), and delta (B.1.617.2), respectively (n = 3 replicates).

    Journal: medRxiv

    Article Title: A Thermostable Cas12b from Brevibacillus Leverages One-pot Detection of SARS-CoV-2 Variants of Concern

    doi: 10.1101/2021.10.15.21265066

    Figure Lengend Snippet: BrCas12b characterization and one-pot specificity sensitivity testing. ( a ) Schematic of binary complex illustrating cleavage pattern of dsDNA target. ( b ) Temperature-dependent in-vitro cleavage assay of AacCas12b, AapCas12b, and BrCas12b targeting more restrictive PAM TTTG. 125 nM sgRNA:100 nM Cas12b:7 nM dsDNA was combined in 1X NEBuffer 2.1. The experiment was repeated (n = 2) with similar results. ( c ) Michaelis-Menten Kinetics of BrCas12b trans-cleavage at 62°C. ( d ) Differential scanning fluorimetry of AacCas12b, AapCas12b, and BrCas12b complexes. (e) Multiplexing using FITC to detect LAMP amplification and HEX to monitor trans-cleavage of BrCas12b. ( f ) – ( i ) Detection capability of BrCas12b via trans-cleavage against AacCas12b and AapCas12b at various temperatures. Fluorescence kinetics within 30 minutes was monitored using a HEX-based reporter. Shaded regions represent standard deviation (n = 3 replicates). STOPCovid LAMP primers were used in (f) to compare performance with AapCas12b. A different set of LAMP primers (DETECTR) with optimal performance at a temperature higher than 60°C were used in (g), (h), and (i). (+) denotes the presence of the SARS-CoV-2 genomic RNA control, and (-) signifies the non-template control (NTC). ( j ) – ( m ) Specificity testing using a pair of sgRNA and LAMP primers targeting SARS-CoV-2 variants (n = 3 replicates). ( n ) – ( q ) Limit of detection on N gene and S gene targeting alpha (B.1.1.7), beta (B.1.352), and delta (B.1.617.2), respectively (n = 3 replicates).

    Article Snippet: In short, BrCas12b, sgRNA, and dsDNA activator were combined to a final concentration of 100 nM: 125 nM: 1 nM respectively in 1x NEBuffer 2.1 (New England Biolabs) and incubated at 62°C for 30 minutes.

    Techniques: In Vitro, Cleavage Assay, Multiplexing, Amplification, Fluorescence, Standard Deviation

    BrCas12b characterisation and one-pot specificity    sensitivity testing. (a) Schematic of binary complex illustrating cleavage pattern of dsDNA target. (b) Temperature-dependent  in-vitro  cleavage assay of AacCas12b, AapCas12b, and BrCas12b targeting more restrictive PAM TTTG. 125 nM sgRNA:100 nM Cas12b:7 nM dsDNA was combined in 1X NEBuffer 2·1. The experiment was repeated ( n  = 2 biological replicates) with similar results. (c) Michaelis-Menten Kinetics of BrCas12b trans-cleavage at 62 °C. (d) Differential scanning fluorimetry of apo AacCas12b, apo AapCas12b, and apo BrCas12b and their corresponding Cas12b:sgRNA complexes. (e) Multiplexing using FITC to detect LAMP amplification and HEX to monitor trans-cleavage of BrCas12b. (f)–(i) Detection capability of BrCas12b via trans-cleavage against AacCas12b and AapCas12b at various temperatures. Fluorescence kinetics within 30 min was monitored using HEX-based reporter. Shaded regions represent standard deviation ( n  = 3 biological replicates). STOPCovid LAMP primers were used in (f) to compare performance with AapCas12b. A different set of LAMP primers (DETECTR) with optimal performance at temperature higher than 60 °C were used in (g), (h), and (i). (+) denotes the presence of the SARS-CoV-2 genomic RNA control, and (-) signifies the non-template control (NTC). (j)–(n) Specificity testing using a pair of sgRNA and LAMP primers targeting SARS-CoV-2 variants of concern ( n  = 3 biological replicates). (o) – (t) Limit of detection of non-N gene targeting Alpha (B.1.1.7), Beta (B.1.352), Gamma (P1), Delta (B.1.617.2), Omicron (B.1.1.529) and N gene, respectively ( n  = 3 biological replicates).

    Journal: EBioMedicine

    Article Title: A thermostable Cas12b from Brevibacillus leverages one-pot discrimination of SARS-CoV-2 variants of concern

    doi: 10.1016/j.ebiom.2022.103926

    Figure Lengend Snippet: BrCas12b characterisation and one-pot specificity sensitivity testing. (a) Schematic of binary complex illustrating cleavage pattern of dsDNA target. (b) Temperature-dependent in-vitro cleavage assay of AacCas12b, AapCas12b, and BrCas12b targeting more restrictive PAM TTTG. 125 nM sgRNA:100 nM Cas12b:7 nM dsDNA was combined in 1X NEBuffer 2·1. The experiment was repeated ( n  = 2 biological replicates) with similar results. (c) Michaelis-Menten Kinetics of BrCas12b trans-cleavage at 62 °C. (d) Differential scanning fluorimetry of apo AacCas12b, apo AapCas12b, and apo BrCas12b and their corresponding Cas12b:sgRNA complexes. (e) Multiplexing using FITC to detect LAMP amplification and HEX to monitor trans-cleavage of BrCas12b. (f)–(i) Detection capability of BrCas12b via trans-cleavage against AacCas12b and AapCas12b at various temperatures. Fluorescence kinetics within 30 min was monitored using HEX-based reporter. Shaded regions represent standard deviation ( n  = 3 biological replicates). STOPCovid LAMP primers were used in (f) to compare performance with AapCas12b. A different set of LAMP primers (DETECTR) with optimal performance at temperature higher than 60 °C were used in (g), (h), and (i). (+) denotes the presence of the SARS-CoV-2 genomic RNA control, and (-) signifies the non-template control (NTC). (j)–(n) Specificity testing using a pair of sgRNA and LAMP primers targeting SARS-CoV-2 variants of concern ( n  = 3 biological replicates). (o) – (t) Limit of detection of non-N gene targeting Alpha (B.1.1.7), Beta (B.1.352), Gamma (P1), Delta (B.1.617.2), Omicron (B.1.1.529) and N gene, respectively ( n  = 3 biological replicates).

    Article Snippet: In short, BrCas12b, sgRNA, and dsDNA activator were combined to a final concentration of 100 nM : 125 nM : 1 nM, respectively in 1x NEBuffer 2·1 (New England Biolabs) and incubated at 62 °C for 30 min. HEX-polyT-Quencher reporter (FQ) at various concentrations (10 nM, 100 nM, 200 nM, 500 nM, 1 µM, and 2 µM) was added to the reaction mixture containing Cas12b trans-activated complex; and the entire reaction was then immediately transferred to a plate reader.

    Techniques: In Vitro, Cleavage Assay, Multiplexing, Amplification, Fluorescence, Standard Deviation