bst2 0 warmstart polymerase  (New England Biolabs)


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    Name:
    Bst 2 0 WarmStart DNA Polymerase
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    Bst 2 0 WarmStart DNA Polymerase 8 000 units
    Catalog Number:
    m0538l
    Price:
    312
    Size:
    8 000 units
    Category:
    Thermostable DNA Polymerases
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    New England Biolabs bst2 0 warmstart polymerase
    Bst 2 0 WarmStart DNA Polymerase
    Bst 2 0 WarmStart DNA Polymerase 8 000 units
    https://www.bioz.com/result/bst2 0 warmstart polymerase/product/New England Biolabs
    Average 95 stars, based on 62 article reviews
    Price from $9.99 to $1999.99
    bst2 0 warmstart polymerase - by Bioz Stars, 2020-02
    95/100 stars

    Images

    1) Product Images from "NGS-based deep bisulfite sequencing"

    Article Title: NGS-based deep bisulfite sequencing

    Journal: MethodsX

    doi: 10.1016/j.mex.2015.11.008

    Overall scheme for NGS-based deep bisulfite sequencing. (A) The entire procedure of the NGS-based deep bisulfite sequencing protocol is shown as a flow chart. (B) The adaptor ligation step for the current protocol has adopted one strategy, in which the added PCR products and two adaptors are ligated through two stepwise incubations. Since both adaptors lack the phosphate group at their 5′-ends, the ligation reaction by T4 at 25 °C occurs between only one strand of the adaptors and the PCR products. In this case, the phosphate groups are derived from the 5′-end of the PCR products. At 65 °C, the activated Bst2.0 WarmStart polymerase extends and displaces the other unligated strand from the partially joined products. The * symbol indicates the phosphate group at the 5′-end of the end-repaired PCR products. (C) The sequences of Ion Torrent P1 and A adaptors are shown with different colors to indicate the key, barcode and spacer regions. The * symbol indicates a phosphothiate bonding between two nucleotides, which protects the duplex adaptor from being digested by the exonuclease activity of DNA polymerases.
    Figure Legend Snippet: Overall scheme for NGS-based deep bisulfite sequencing. (A) The entire procedure of the NGS-based deep bisulfite sequencing protocol is shown as a flow chart. (B) The adaptor ligation step for the current protocol has adopted one strategy, in which the added PCR products and two adaptors are ligated through two stepwise incubations. Since both adaptors lack the phosphate group at their 5′-ends, the ligation reaction by T4 at 25 °C occurs between only one strand of the adaptors and the PCR products. In this case, the phosphate groups are derived from the 5′-end of the PCR products. At 65 °C, the activated Bst2.0 WarmStart polymerase extends and displaces the other unligated strand from the partially joined products. The * symbol indicates the phosphate group at the 5′-end of the end-repaired PCR products. (C) The sequences of Ion Torrent P1 and A adaptors are shown with different colors to indicate the key, barcode and spacer regions. The * symbol indicates a phosphothiate bonding between two nucleotides, which protects the duplex adaptor from being digested by the exonuclease activity of DNA polymerases.

    Techniques Used: Next-Generation Sequencing, Methylation Sequencing, Flow Cytometry, Ligation, Polymerase Chain Reaction, Derivative Assay, Activity Assay

    2) Product Images from "Diagnosis of Brugian Filariasis by Loop-Mediated Isothermal Amplification"

    Article Title: Diagnosis of Brugian Filariasis by Loop-Mediated Isothermal Amplification

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0001948

    Species-specificity of Hha I LAMP assay. (A) Each curve represents the calculated average of triplicate turbidity curves generated with various genomic DNAs (0. 1 ng) using Bst 2.0 DNA polymerase without loop primers. Turbidity was observed using B. malayi or B. timori DNA. (B) As a positive control, an actin gene fragment was PCR amplified from B. malayi (Bma), D. immitis (Dim), O. volvulus (Ovo), the mosquito Aedes albopictus (Aal), W. bancrofti (Wba), human (Hsa) and B. timori (Bti) DNAs using degenerate primers. Agarose gel showing amplification of a 244 bp fragment of the actin gene. The 100 bp DNA Ladder (New England Biolabs) was used as the molecular weight marker (MWM). Water was used in the non-template controls (NTC) in (A) and (B).
    Figure Legend Snippet: Species-specificity of Hha I LAMP assay. (A) Each curve represents the calculated average of triplicate turbidity curves generated with various genomic DNAs (0. 1 ng) using Bst 2.0 DNA polymerase without loop primers. Turbidity was observed using B. malayi or B. timori DNA. (B) As a positive control, an actin gene fragment was PCR amplified from B. malayi (Bma), D. immitis (Dim), O. volvulus (Ovo), the mosquito Aedes albopictus (Aal), W. bancrofti (Wba), human (Hsa) and B. timori (Bti) DNAs using degenerate primers. Agarose gel showing amplification of a 244 bp fragment of the actin gene. The 100 bp DNA Ladder (New England Biolabs) was used as the molecular weight marker (MWM). Water was used in the non-template controls (NTC) in (A) and (B).

    Techniques Used: Lamp Assay, Generated, Positive Control, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Molecular Weight, Marker

    Sensitivity of Hha I LAMP assay. Ten-fold serial dilutions of B. malayi genomic DNA amplified with the Hha I primer set alone (A) or in the presence of loop primers (B) with Bst DNA polymerase, large fragment (wt Bst LF), Bst 2.0 DNA polymerase ( Bst 2.0) and Bst 2.0 WarmStart DNA polymerase ( Bst 2.0 WS). Data points represent the average of three samples and the error bars represent the standard deviation at each point. For each enzyme, the average threshold time, defined as the time at which the change in turbidity over time (dT/dt) reaches a value of 0.1, is plotted against the amount of starting material. (C) UV detection (365 nm) of products generated within 60 minutes using Bst 2.0 in the presence of loop primers and Fluorescent Detection Reagent. The amount of starting material in ng is shown below the photograph. Positive samples fluoresce green while negative samples remain dark.
    Figure Legend Snippet: Sensitivity of Hha I LAMP assay. Ten-fold serial dilutions of B. malayi genomic DNA amplified with the Hha I primer set alone (A) or in the presence of loop primers (B) with Bst DNA polymerase, large fragment (wt Bst LF), Bst 2.0 DNA polymerase ( Bst 2.0) and Bst 2.0 WarmStart DNA polymerase ( Bst 2.0 WS). Data points represent the average of three samples and the error bars represent the standard deviation at each point. For each enzyme, the average threshold time, defined as the time at which the change in turbidity over time (dT/dt) reaches a value of 0.1, is plotted against the amount of starting material. (C) UV detection (365 nm) of products generated within 60 minutes using Bst 2.0 in the presence of loop primers and Fluorescent Detection Reagent. The amount of starting material in ng is shown below the photograph. Positive samples fluoresce green while negative samples remain dark.

    Techniques Used: Lamp Assay, Amplification, Standard Deviation, Generated

    Hha I LAMP assay for the detection of B. malayi infected blood samples. A set of serial dilutions (two-fold) of microfilariae in blood was prepared and DNA was isolated from each dilution. Three experiments were performed using a different but overlapping range of DNA dilutions. One µl of DNA from each dilution was used in LAMP reactions with Bst 2.0 DNA polymerase. Samples from each experimental set-up were performed in triplicate (experiments 1 and 2) or duplicate (experiment 3). Average threshold times and standard deviations were plotted against the approximate number of mf/µl DNA solution.
    Figure Legend Snippet: Hha I LAMP assay for the detection of B. malayi infected blood samples. A set of serial dilutions (two-fold) of microfilariae in blood was prepared and DNA was isolated from each dilution. Three experiments were performed using a different but overlapping range of DNA dilutions. One µl of DNA from each dilution was used in LAMP reactions with Bst 2.0 DNA polymerase. Samples from each experimental set-up were performed in triplicate (experiments 1 and 2) or duplicate (experiment 3). Average threshold times and standard deviations were plotted against the approximate number of mf/µl DNA solution.

    Techniques Used: Lamp Assay, Infection, Isolation

    3) Product Images from "NGS-based deep bisulfite sequencing"

    Article Title: NGS-based deep bisulfite sequencing

    Journal: MethodsX

    doi: 10.1016/j.mex.2015.11.008

    Overall scheme for NGS-based deep bisulfite sequencing. (A) The entire procedure of the NGS-based deep bisulfite sequencing protocol is shown as a flow chart. (B) The adaptor ligation step for the current protocol has adopted one strategy, in which the added PCR products and two adaptors are ligated through two stepwise incubations. Since both adaptors lack the phosphate group at their 5′-ends, the ligation reaction by T4 at 25 °C occurs between only one strand of the adaptors and the PCR products. In this case, the phosphate groups are derived from the 5′-end of the PCR products. At 65 °C, the activated Bst2.0 WarmStart polymerase extends and displaces the other unligated strand from the partially joined products. The * symbol indicates the phosphate group at the 5′-end of the end-repaired PCR products. (C) The sequences of Ion Torrent P1 and A adaptors are shown with different colors to indicate the key, barcode and spacer regions. The * symbol indicates a phosphothiate bonding between two nucleotides, which protects the duplex adaptor from being digested by the exonuclease activity of DNA polymerases.
    Figure Legend Snippet: Overall scheme for NGS-based deep bisulfite sequencing. (A) The entire procedure of the NGS-based deep bisulfite sequencing protocol is shown as a flow chart. (B) The adaptor ligation step for the current protocol has adopted one strategy, in which the added PCR products and two adaptors are ligated through two stepwise incubations. Since both adaptors lack the phosphate group at their 5′-ends, the ligation reaction by T4 at 25 °C occurs between only one strand of the adaptors and the PCR products. In this case, the phosphate groups are derived from the 5′-end of the PCR products. At 65 °C, the activated Bst2.0 WarmStart polymerase extends and displaces the other unligated strand from the partially joined products. The * symbol indicates the phosphate group at the 5′-end of the end-repaired PCR products. (C) The sequences of Ion Torrent P1 and A adaptors are shown with different colors to indicate the key, barcode and spacer regions. The * symbol indicates a phosphothiate bonding between two nucleotides, which protects the duplex adaptor from being digested by the exonuclease activity of DNA polymerases.

    Techniques Used: Next-Generation Sequencing, Methylation Sequencing, Flow Cytometry, Ligation, Polymerase Chain Reaction, Derivative Assay, Activity Assay

    4) Product Images from "A Novel Single-Nucleotide Polymorphism Loop Mediated Isothermal Amplification Assay for Detection of Artemisinin-Resistant Plasmodium falciparum Malaria"

    Article Title: A Novel Single-Nucleotide Polymorphism Loop Mediated Isothermal Amplification Assay for Detection of Artemisinin-Resistant Plasmodium falciparum Malaria

    Journal: Open Forum Infectious Diseases

    doi: 10.1093/ofid/ofy011

    Single-nucleotide polymorphism loop mediated isothermal amplification (SNP-LAMP) of mutant Y580 kelch13 from laboratory strain MRA 1240 (Y580, gray bars) versus wild-type MRA 1236 (C580, black bars). The SNP-LAMP was performed using Bst 2.0 WarmStart DNA polymerase at 63 o C with primer set 31. Turbidity was measured at 600-nm wavelength by NanoDrop at different reaction time points: (a) 50 minutes, (b) 55 minutes, (c) 60 minutes, (d) 65 minutes, (e) 70 minutes, and (f) 75 minutes.
    Figure Legend Snippet: Single-nucleotide polymorphism loop mediated isothermal amplification (SNP-LAMP) of mutant Y580 kelch13 from laboratory strain MRA 1240 (Y580, gray bars) versus wild-type MRA 1236 (C580, black bars). The SNP-LAMP was performed using Bst 2.0 WarmStart DNA polymerase at 63 o C with primer set 31. Turbidity was measured at 600-nm wavelength by NanoDrop at different reaction time points: (a) 50 minutes, (b) 55 minutes, (c) 60 minutes, (d) 65 minutes, (e) 70 minutes, and (f) 75 minutes.

    Techniques Used: Amplification, Mutagenesis

    Single-nucleotide polymorphism loop mediated isothermal amplification of mutant Y580 kelch13 from laboratory strain MRA 1240 (Y580, gray bars) versus wild-type MRA 1236 (C580, black bars) using Bst 2.0 WarmStart DNA polymerase at (a) 59°C, (b) 61°C, (c) 63°C, and (d) 65°C for 60 minutes. The template used was a polymerase chain reaction product of the kelch13 propellor domain with primer set 31.
    Figure Legend Snippet: Single-nucleotide polymorphism loop mediated isothermal amplification of mutant Y580 kelch13 from laboratory strain MRA 1240 (Y580, gray bars) versus wild-type MRA 1236 (C580, black bars) using Bst 2.0 WarmStart DNA polymerase at (a) 59°C, (b) 61°C, (c) 63°C, and (d) 65°C for 60 minutes. The template used was a polymerase chain reaction product of the kelch13 propellor domain with primer set 31.

    Techniques Used: Amplification, Mutagenesis, Polymerase Chain Reaction

    Amplification time (minutes) required for a positive call by 3 different enzymes using single-nucleotide polymorphism loop mediated isothermal amplification for Plasmodium falciparum -culture spiked whole blood: (a) GspSSD2 enzyme (63 o C); (b) Bst 2.0 WarmStart DNA polymerase (63 o C). These studies were performed on the CFX-96 detection system.
    Figure Legend Snippet: Amplification time (minutes) required for a positive call by 3 different enzymes using single-nucleotide polymorphism loop mediated isothermal amplification for Plasmodium falciparum -culture spiked whole blood: (a) GspSSD2 enzyme (63 o C); (b) Bst 2.0 WarmStart DNA polymerase (63 o C). These studies were performed on the CFX-96 detection system.

    Techniques Used: Amplification

    5) Product Images from "Advanced uracil DNA glycosylase-supplemented real-time reverse transcription loop-mediated isothermal amplification (UDG-rRT-LAMP) method for universal and specific detection of Tembusu virus"

    Article Title: Advanced uracil DNA glycosylase-supplemented real-time reverse transcription loop-mediated isothermal amplification (UDG-rRT-LAMP) method for universal and specific detection of Tembusu virus

    Journal: Scientific Reports

    doi: 10.1038/srep27605

    Schematic diagram showing the mechanism of the uracil DNA glycosylase-supplemented real-time reverse-transcription loop-mediated isothermal amplification (UDG-rRT-LAMP) assay. Blue LAMP amplicons represent non-dUTP-incorporated DNA and yellow LAMP amplicons represent dUTP-incorporated DNA. The red enzyme represents Bst 2.0 WarmStart ® DNA polymerase and the blue enzyme represents UDG. UDG-rRT-LAMP eliminates carryover contamination via two steps. ( A ) The first step of the incorporation of dUTP in all LAMP amplicons. ( B ) The second step of UDG-based elimination of carryover contaminants by specifically cutting the LAMP amplicon DNA at the 5′ side of the dUTP-incorporated templates from previous LAMP reactions while having no effect on non-dUTP-incorporated DNA and RNA templates. During the RT-LAMP reaction, the digested contaminants are degraded into small fragments, and UDG is inactivated at approximately 63 °C, ensuring that only the RNA template is amplified.
    Figure Legend Snippet: Schematic diagram showing the mechanism of the uracil DNA glycosylase-supplemented real-time reverse-transcription loop-mediated isothermal amplification (UDG-rRT-LAMP) assay. Blue LAMP amplicons represent non-dUTP-incorporated DNA and yellow LAMP amplicons represent dUTP-incorporated DNA. The red enzyme represents Bst 2.0 WarmStart ® DNA polymerase and the blue enzyme represents UDG. UDG-rRT-LAMP eliminates carryover contamination via two steps. ( A ) The first step of the incorporation of dUTP in all LAMP amplicons. ( B ) The second step of UDG-based elimination of carryover contaminants by specifically cutting the LAMP amplicon DNA at the 5′ side of the dUTP-incorporated templates from previous LAMP reactions while having no effect on non-dUTP-incorporated DNA and RNA templates. During the RT-LAMP reaction, the digested contaminants are degraded into small fragments, and UDG is inactivated at approximately 63 °C, ensuring that only the RNA template is amplified.

    Techniques Used: Amplification, Lamp Assay

    6) Product Images from "Unusual isothermal multimerization and amplification by the strand-displacing DNA polymerases with reverse transcription activities"

    Article Title: Unusual isothermal multimerization and amplification by the strand-displacing DNA polymerases with reverse transcription activities

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-13324-0

    Verification of UIMA using different DNA polymerases. All reactions shared the same primer (RL) and template (F*R*) and were incubated for 180 min. The sequences of RL and F*R* were shown in Table S1 . ( A ) Real-time fluorescence change in reactions using a series of Bst DNA polymerases ( Bst LF, Bst 2.0, Bst 2.0 WS, and Bst 3.0) at 63 °C. No-primer controls (NPCs) were shown in Fig. S5 . ( B ) Real-time fluorescence change in reactions using non- Bst polymerases (Bsm, BcaBEST, Vent(exo-), and z-Taq) at 63 °C. No-primer controls (NPCs) were shown in Fig. S5 . ( C ) Temperature gradients assay for the products of reactions using the polymerases with negative results in ( B ). The products were analyzed by 2.5% agarose gel electrophoresis. NTC and NPC for Bsm were performed at 56 °C. NTCs and NPCs for Vent (exo-) and z-Taq were performed at 63 °C. The groping of gels cropped from different gels. Exposure time is 5 s. ( D ) Temperature gradients assay for the products of reactions using the polymerases of Klenow(exo-) and Klenow. The products were analyzed by 2.5% agarose gel electrophoresis. Their NTCs and NPCs were performed at 43 °C. M1 and M2: DNA Marker. NTC: no-target control; NPC: no-primer control. The groping of gels cropped from different gels. Exposure time is 5 s. The full-length gels are presented in Supplementary Figure S7 .
    Figure Legend Snippet: Verification of UIMA using different DNA polymerases. All reactions shared the same primer (RL) and template (F*R*) and were incubated for 180 min. The sequences of RL and F*R* were shown in Table S1 . ( A ) Real-time fluorescence change in reactions using a series of Bst DNA polymerases ( Bst LF, Bst 2.0, Bst 2.0 WS, and Bst 3.0) at 63 °C. No-primer controls (NPCs) were shown in Fig. S5 . ( B ) Real-time fluorescence change in reactions using non- Bst polymerases (Bsm, BcaBEST, Vent(exo-), and z-Taq) at 63 °C. No-primer controls (NPCs) were shown in Fig. S5 . ( C ) Temperature gradients assay for the products of reactions using the polymerases with negative results in ( B ). The products were analyzed by 2.5% agarose gel electrophoresis. NTC and NPC for Bsm were performed at 56 °C. NTCs and NPCs for Vent (exo-) and z-Taq were performed at 63 °C. The groping of gels cropped from different gels. Exposure time is 5 s. ( D ) Temperature gradients assay for the products of reactions using the polymerases of Klenow(exo-) and Klenow. The products were analyzed by 2.5% agarose gel electrophoresis. Their NTCs and NPCs were performed at 43 °C. M1 and M2: DNA Marker. NTC: no-target control; NPC: no-primer control. The groping of gels cropped from different gels. Exposure time is 5 s. The full-length gels are presented in Supplementary Figure S7 .

    Techniques Used: Incubation, Fluorescence, Agarose Gel Electrophoresis, Marker

    Real-time fluorescence and electrophoresis analysis of UIMA. All reactions shared the same primer (RL) or template (F*R*), and were incubated for 180 min. The sequences of RL and F*R* were shown in Table S1 . ( A ) The results of real-time fluorescence obtained from the reactions that contained 10 nM template, 1.6 μM primer, and 3.2 U Bst WS DNA polymerase at 63 °C for 180 min. Each test was in triplicate. ( B ) Time course of the UIMA assay. 2.5% agarose gel electrophoresis shows the products of UIMA. The assay time was varied from 30–120 minutes as indicated above each lane. M1 and M2: DNA marker; NEC, NTC and NPC were all incubated for 120 min. Exposure time is 5 s. ( C ) Extension status of template and primer. Template and primer were labeled with the FAM fluorophore. 1: reaction with FAM-labeled primer and template but no Bst ; 2: with FAM-labeled primer, non-labeled template, and Bst ; 3: with FAM-labeled primer and template and Bst ; 4: with FAM-labeled primer and Bst but no template; 5: with non-labeled primer and template, and Bst ; 6: with non-labeled primer, FAM-labeled template, and Bst ; 7: with non-labeled primer and Bst but no template; 8: with non-labeled template and Bst but no primer; 9: with FAM-labeled template and Bst but no primer. All the reactions were incubated at 63 °C for 180 min. Their products were analyzed by 17% denatured polyacrylamide gel electrophoresis (DPAGE). NEC (no-enzyme control): the reaction without Bst 2.0 WS DNA polymerase. NTC (no-template control): control reaction just lacked the template; NPC (no-primer control): control reaction lacked primer RL. Horizontal arrows denoted the 5′-3′ direction of sequences. Exposure time is 5 s. The full-length gels are presented in Supplementary Figure S1 .
    Figure Legend Snippet: Real-time fluorescence and electrophoresis analysis of UIMA. All reactions shared the same primer (RL) or template (F*R*), and were incubated for 180 min. The sequences of RL and F*R* were shown in Table S1 . ( A ) The results of real-time fluorescence obtained from the reactions that contained 10 nM template, 1.6 μM primer, and 3.2 U Bst WS DNA polymerase at 63 °C for 180 min. Each test was in triplicate. ( B ) Time course of the UIMA assay. 2.5% agarose gel electrophoresis shows the products of UIMA. The assay time was varied from 30–120 minutes as indicated above each lane. M1 and M2: DNA marker; NEC, NTC and NPC were all incubated for 120 min. Exposure time is 5 s. ( C ) Extension status of template and primer. Template and primer were labeled with the FAM fluorophore. 1: reaction with FAM-labeled primer and template but no Bst ; 2: with FAM-labeled primer, non-labeled template, and Bst ; 3: with FAM-labeled primer and template and Bst ; 4: with FAM-labeled primer and Bst but no template; 5: with non-labeled primer and template, and Bst ; 6: with non-labeled primer, FAM-labeled template, and Bst ; 7: with non-labeled primer and Bst but no template; 8: with non-labeled template and Bst but no primer; 9: with FAM-labeled template and Bst but no primer. All the reactions were incubated at 63 °C for 180 min. Their products were analyzed by 17% denatured polyacrylamide gel electrophoresis (DPAGE). NEC (no-enzyme control): the reaction without Bst 2.0 WS DNA polymerase. NTC (no-template control): control reaction just lacked the template; NPC (no-primer control): control reaction lacked primer RL. Horizontal arrows denoted the 5′-3′ direction of sequences. Exposure time is 5 s. The full-length gels are presented in Supplementary Figure S1 .

    Techniques Used: Fluorescence, Electrophoresis, Incubation, Agarose Gel Electrophoresis, Marker, Labeling, Polyacrylamide Gel Electrophoresis

    7) Product Images from "Development of a loop-mediated isothermal amplification (LAMP) assay for rapid screening of ticks and fleas for spotted fever group rickettsia"

    Article Title: Development of a loop-mediated isothermal amplification (LAMP) assay for rapid screening of ticks and fleas for spotted fever group rickettsia

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0192331

    Specificity of SFGR-LAMP assay using Bst 2.0 WarmStart DNA polymerase. (A). Colorimetric visual detection of hydroxynaphol blue-based SFGR-LAMP reaction products inspected by the naked eye. The color changes from light blue in the positive reactions to dark blue to purple in the negative reactions. (B). Agarose gel electrophoresis. Lane 1: 100Kbp molecular ladder; 2: water control; 3: positive control ( R . rickettsii ); 4: R . africae ; 5: R . amblyommatis ; 6: R . conorii ; 7: R . felis ; 8: R . montanensis ; 9: R . parkeri ; 10: R . rickettsii ; 11: R . typhi ; 12: E . chaffeensis ; 13: A . phagocytophilum ; 14: C . burnetii ; 15: E . coli ; 16: S . typhi .
    Figure Legend Snippet: Specificity of SFGR-LAMP assay using Bst 2.0 WarmStart DNA polymerase. (A). Colorimetric visual detection of hydroxynaphol blue-based SFGR-LAMP reaction products inspected by the naked eye. The color changes from light blue in the positive reactions to dark blue to purple in the negative reactions. (B). Agarose gel electrophoresis. Lane 1: 100Kbp molecular ladder; 2: water control; 3: positive control ( R . rickettsii ); 4: R . africae ; 5: R . amblyommatis ; 6: R . conorii ; 7: R . felis ; 8: R . montanensis ; 9: R . parkeri ; 10: R . rickettsii ; 11: R . typhi ; 12: E . chaffeensis ; 13: A . phagocytophilum ; 14: C . burnetii ; 15: E . coli ; 16: S . typhi .

    Techniques Used: Lamp Assay, Agarose Gel Electrophoresis, Positive Control

    8) Product Images from "A Novel Single-Nucleotide Polymorphism Loop Mediated Isothermal Amplification Assay for Detection of Artemisinin-Resistant Plasmodium falciparum Malaria"

    Article Title: A Novel Single-Nucleotide Polymorphism Loop Mediated Isothermal Amplification Assay for Detection of Artemisinin-Resistant Plasmodium falciparum Malaria

    Journal: Open Forum Infectious Diseases

    doi: 10.1093/ofid/ofy011

    Amplification time (minutes) required for a positive call by 3 different enzymes using single-nucleotide polymorphism loop mediated isothermal amplification for Plasmodium falciparum -culture spiked whole blood: (a) GspSSD2 enzyme (63 o C); (b) Bst 2.0 WarmStart DNA polymerase (63 o C). These studies were performed on the CFX-96 detection system.
    Figure Legend Snippet: Amplification time (minutes) required for a positive call by 3 different enzymes using single-nucleotide polymorphism loop mediated isothermal amplification for Plasmodium falciparum -culture spiked whole blood: (a) GspSSD2 enzyme (63 o C); (b) Bst 2.0 WarmStart DNA polymerase (63 o C). These studies were performed on the CFX-96 detection system.

    Techniques Used: Amplification

    Single-nucleotide polymorphism loop mediated isothermal amplification of mutant Y580 kelch13 from laboratory strain MRA 1240 (Y580, gray bars) versus wild-type MRA 1236 (C580, black bars) using Bst 2.0 WarmStart DNA polymerase at (a) 59°C, (b) 61°C, (c) 63°C, and (d) 65°C for 60 minutes. The template used was a polymerase chain reaction product of the kelch13 propellor domain with primer set 31.
    Figure Legend Snippet: Single-nucleotide polymorphism loop mediated isothermal amplification of mutant Y580 kelch13 from laboratory strain MRA 1240 (Y580, gray bars) versus wild-type MRA 1236 (C580, black bars) using Bst 2.0 WarmStart DNA polymerase at (a) 59°C, (b) 61°C, (c) 63°C, and (d) 65°C for 60 minutes. The template used was a polymerase chain reaction product of the kelch13 propellor domain with primer set 31.

    Techniques Used: Amplification, Mutagenesis, Polymerase Chain Reaction

    Single-nucleotide polymorphism loop mediated isothermal amplification (SNP-LAMP) of mutant Y580 kelch13 from laboratory strain MRA 1240 (Y580, gray bars) versus wild-type MRA 1236 (C580, black bars). The SNP-LAMP was performed using Bst 2.0 WarmStart DNA polymerase at 63 o C with primer set 31. Turbidity was measured at 600-nm wavelength by NanoDrop at different reaction time points: (a) 50 minutes, (b) 55 minutes, (c) 60 minutes, (d) 65 minutes, (e) 70 minutes, and (f) 75 minutes.
    Figure Legend Snippet: Single-nucleotide polymorphism loop mediated isothermal amplification (SNP-LAMP) of mutant Y580 kelch13 from laboratory strain MRA 1240 (Y580, gray bars) versus wild-type MRA 1236 (C580, black bars). The SNP-LAMP was performed using Bst 2.0 WarmStart DNA polymerase at 63 o C with primer set 31. Turbidity was measured at 600-nm wavelength by NanoDrop at different reaction time points: (a) 50 minutes, (b) 55 minutes, (c) 60 minutes, (d) 65 minutes, (e) 70 minutes, and (f) 75 minutes.

    Techniques Used: Amplification, Mutagenesis

    9) Product Images from "Rapid Multiplex Small DNA Sequencing on the MinION Nanopore Sequencing Platform"

    Article Title: Rapid Multiplex Small DNA Sequencing on the MinION Nanopore Sequencing Platform

    Journal: G3: Genes|Genomes|Genetics

    doi: 10.1534/g3.118.200087

    Optimization of MinION library preparation. A). Optimization of ligation condition for TA ligation and 6-bp sticky-end ligation. Condition 1. The manufacturer’s suggested condition; 2. the condition reported before ( wei and williams 2016 ); 3-5. the conditions with addition of 6%, 9%, and 12% enhancer mix. Efficiencies of 6-bp ligation were estimated using a pair of adaptor MP1-6bp and ME-6bp carrying complementary 6-bp sticky ends. Efficiencies of TA ligation were estimated using a pair of adaptor MP1-T and ME-A carrying complementary 3′T and 3′A overhangs. B). Titration experiment of Native Barcode (NB) adapter. 6.5ng, 9.8ng, 13ng of NB adapters were added in to the 1-step ligation reaction which contains the same amount of dA-tailed DNA and MP1-6bp adapter. The expected final products with 2-end ligated to a barcode and MP1-6bp adapter were marked in bold. The products separated on gel were also illustrated in cartoons (MP1-6bp adapter: green; NB adapter: blue; dA-tailed DNA: purple). C). Optimization of end-repair/dA-tailling condition. Lane 1, the input 434bp control fragment; lane 2, manufacturer’s recommended protocol; lane 3. the optimized condition; lane 4. The optimized condition with supplementation of Bst 2.0 WarmStart Polymerase. The expected products with 2-end ligated to an adapter were marked in bold and the products separated on gel were also illustrated in cartoons (434bp dA-tailed DNA: purple; MP1-T adapter: dark green). D). Optimization of AMPure XP bead purification by changing the volume of bead. 100 ng 50bp ladder and 2 pmole 204bp control fragment were used as input, and subjected to onefold, 0.65-fold, 0.sixfold, 0.55-fold AMPure XP bead purification. The expected products are bands > 500 bp, and it’s marked in bold E). Optimization of AMPure XP bead purification by adjusting the concentration of PEG in wash buffer. 100 ng 50bp ladder and 2 pmole 204bp control fragment were used as input, and subjected to 0.62-fold AMPure XP bead purification using wash buffer containing 10%, 9%, 8.5% and 8% PEG. The expected products are bands > 500 bp, and it’s marked in bold. F). Optimization of tethering condition. Lane 1-5: 1µL BAM adapter with 0-4µL ELB buffer after 3min incubation at 37°C. The expected tethered BAM adapter was marked in bold. The products separated on gel were illustrated in cartoons (BAM adapter: gray; tether: pink-black).
    Figure Legend Snippet: Optimization of MinION library preparation. A). Optimization of ligation condition for TA ligation and 6-bp sticky-end ligation. Condition 1. The manufacturer’s suggested condition; 2. the condition reported before ( wei and williams 2016 ); 3-5. the conditions with addition of 6%, 9%, and 12% enhancer mix. Efficiencies of 6-bp ligation were estimated using a pair of adaptor MP1-6bp and ME-6bp carrying complementary 6-bp sticky ends. Efficiencies of TA ligation were estimated using a pair of adaptor MP1-T and ME-A carrying complementary 3′T and 3′A overhangs. B). Titration experiment of Native Barcode (NB) adapter. 6.5ng, 9.8ng, 13ng of NB adapters were added in to the 1-step ligation reaction which contains the same amount of dA-tailed DNA and MP1-6bp adapter. The expected final products with 2-end ligated to a barcode and MP1-6bp adapter were marked in bold. The products separated on gel were also illustrated in cartoons (MP1-6bp adapter: green; NB adapter: blue; dA-tailed DNA: purple). C). Optimization of end-repair/dA-tailling condition. Lane 1, the input 434bp control fragment; lane 2, manufacturer’s recommended protocol; lane 3. the optimized condition; lane 4. The optimized condition with supplementation of Bst 2.0 WarmStart Polymerase. The expected products with 2-end ligated to an adapter were marked in bold and the products separated on gel were also illustrated in cartoons (434bp dA-tailed DNA: purple; MP1-T adapter: dark green). D). Optimization of AMPure XP bead purification by changing the volume of bead. 100 ng 50bp ladder and 2 pmole 204bp control fragment were used as input, and subjected to onefold, 0.65-fold, 0.sixfold, 0.55-fold AMPure XP bead purification. The expected products are bands > 500 bp, and it’s marked in bold E). Optimization of AMPure XP bead purification by adjusting the concentration of PEG in wash buffer. 100 ng 50bp ladder and 2 pmole 204bp control fragment were used as input, and subjected to 0.62-fold AMPure XP bead purification using wash buffer containing 10%, 9%, 8.5% and 8% PEG. The expected products are bands > 500 bp, and it’s marked in bold. F). Optimization of tethering condition. Lane 1-5: 1µL BAM adapter with 0-4µL ELB buffer after 3min incubation at 37°C. The expected tethered BAM adapter was marked in bold. The products separated on gel were illustrated in cartoons (BAM adapter: gray; tether: pink-black).

    Techniques Used: Ligation, Titration, Purification, Concentration Assay, Incubation

    10) Product Images from "Rapid Multiplex Small DNA Sequencing on the MinION Nanopore Sequencing Platform"

    Article Title: Rapid Multiplex Small DNA Sequencing on the MinION Nanopore Sequencing Platform

    Journal: G3: Genes|Genomes|Genetics

    doi: 10.1534/g3.118.200087

    Optimization of MinION library preparation. A). Optimization of ligation condition for TA ligation and 6-bp sticky-end ligation. Condition 1. The manufacturer’s suggested condition; 2. the condition reported before ( wei and williams 2016 ); 3-5. the conditions with addition of 6%, 9%, and 12% enhancer mix. Efficiencies of 6-bp ligation were estimated using a pair of adaptor MP1-6bp and ME-6bp carrying complementary 6-bp sticky ends. Efficiencies of TA ligation were estimated using a pair of adaptor MP1-T and ME-A carrying complementary 3′T and 3′A overhangs. B). Titration experiment of Native Barcode (NB) adapter. 6.5ng, 9.8ng, 13ng of NB adapters were added in to the 1-step ligation reaction which contains the same amount of dA-tailed DNA and MP1-6bp adapter. The expected final products with 2-end ligated to a barcode and MP1-6bp adapter were marked in bold. The products separated on gel were also illustrated in cartoons (MP1-6bp adapter: green; NB adapter: blue; dA-tailed DNA: purple). C). Optimization of end-repair/dA-tailling condition. Lane 1, the input 434bp control fragment; lane 2, manufacturer’s recommended protocol; lane 3. the optimized condition; lane 4. The optimized condition with supplementation of Bst 2.0 WarmStart Polymerase. The expected products with 2-end ligated to an adapter were marked in bold and the products separated on gel were also illustrated in cartoons (434bp dA-tailed DNA: purple; MP1-T adapter: dark green). D). Optimization of AMPure XP bead purification by changing the volume of bead. 100 ng 50bp ladder and 2 pmole 204bp control fragment were used as input, and subjected to onefold, 0.65-fold, 0.sixfold, 0.55-fold AMPure XP bead purification. The expected products are bands > 500 bp, and it’s marked in bold E). Optimization of AMPure XP bead purification by adjusting the concentration of PEG in wash buffer. 100 ng 50bp ladder and 2 pmole 204bp control fragment were used as input, and subjected to 0.62-fold AMPure XP bead purification using wash buffer containing 10%, 9%, 8.5% and 8% PEG. The expected products are bands > 500 bp, and it’s marked in bold. F). Optimization of tethering condition. Lane 1-5: 1µL BAM adapter with 0-4µL ELB buffer after 3min incubation at 37°C. The expected tethered BAM adapter was marked in bold. The products separated on gel were illustrated in cartoons (BAM adapter: gray; tether: pink-black).
    Figure Legend Snippet: Optimization of MinION library preparation. A). Optimization of ligation condition for TA ligation and 6-bp sticky-end ligation. Condition 1. The manufacturer’s suggested condition; 2. the condition reported before ( wei and williams 2016 ); 3-5. the conditions with addition of 6%, 9%, and 12% enhancer mix. Efficiencies of 6-bp ligation were estimated using a pair of adaptor MP1-6bp and ME-6bp carrying complementary 6-bp sticky ends. Efficiencies of TA ligation were estimated using a pair of adaptor MP1-T and ME-A carrying complementary 3′T and 3′A overhangs. B). Titration experiment of Native Barcode (NB) adapter. 6.5ng, 9.8ng, 13ng of NB adapters were added in to the 1-step ligation reaction which contains the same amount of dA-tailed DNA and MP1-6bp adapter. The expected final products with 2-end ligated to a barcode and MP1-6bp adapter were marked in bold. The products separated on gel were also illustrated in cartoons (MP1-6bp adapter: green; NB adapter: blue; dA-tailed DNA: purple). C). Optimization of end-repair/dA-tailling condition. Lane 1, the input 434bp control fragment; lane 2, manufacturer’s recommended protocol; lane 3. the optimized condition; lane 4. The optimized condition with supplementation of Bst 2.0 WarmStart Polymerase. The expected products with 2-end ligated to an adapter were marked in bold and the products separated on gel were also illustrated in cartoons (434bp dA-tailed DNA: purple; MP1-T adapter: dark green). D). Optimization of AMPure XP bead purification by changing the volume of bead. 100 ng 50bp ladder and 2 pmole 204bp control fragment were used as input, and subjected to onefold, 0.65-fold, 0.sixfold, 0.55-fold AMPure XP bead purification. The expected products are bands > 500 bp, and it’s marked in bold E). Optimization of AMPure XP bead purification by adjusting the concentration of PEG in wash buffer. 100 ng 50bp ladder and 2 pmole 204bp control fragment were used as input, and subjected to 0.62-fold AMPure XP bead purification using wash buffer containing 10%, 9%, 8.5% and 8% PEG. The expected products are bands > 500 bp, and it’s marked in bold. F). Optimization of tethering condition. Lane 1-5: 1µL BAM adapter with 0-4µL ELB buffer after 3min incubation at 37°C. The expected tethered BAM adapter was marked in bold. The products separated on gel were illustrated in cartoons (BAM adapter: gray; tether: pink-black).

    Techniques Used: Ligation, Titration, Purification, Concentration Assay, Incubation

    11) Product Images from "Development of a loop-mediated isothermal amplification (LAMP) assay for rapid screening of ticks and fleas for spotted fever group rickettsia"

    Article Title: Development of a loop-mediated isothermal amplification (LAMP) assay for rapid screening of ticks and fleas for spotted fever group rickettsia

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0192331

    Specificity of SFGR-LAMP assay using Bst 2.0 WarmStart DNA polymerase. (A). Colorimetric visual detection of hydroxynaphol blue-based SFGR-LAMP reaction products inspected by the naked eye. The color changes from light blue in the positive reactions to dark blue to purple in the negative reactions. (B). Agarose gel electrophoresis. Lane 1: 100Kbp molecular ladder; 2: water control; 3: positive control ( R . rickettsii ); 4: R . africae ; 5: R . amblyommatis ; 6: R . conorii ; 7: R . felis ; 8: R . montanensis ; 9: R . parkeri ; 10: R . rickettsii ; 11: R . typhi ; 12: E . chaffeensis ; 13: A . phagocytophilum ; 14: C . burnetii ; 15: E . coli ; 16: S . typhi .
    Figure Legend Snippet: Specificity of SFGR-LAMP assay using Bst 2.0 WarmStart DNA polymerase. (A). Colorimetric visual detection of hydroxynaphol blue-based SFGR-LAMP reaction products inspected by the naked eye. The color changes from light blue in the positive reactions to dark blue to purple in the negative reactions. (B). Agarose gel electrophoresis. Lane 1: 100Kbp molecular ladder; 2: water control; 3: positive control ( R . rickettsii ); 4: R . africae ; 5: R . amblyommatis ; 6: R . conorii ; 7: R . felis ; 8: R . montanensis ; 9: R . parkeri ; 10: R . rickettsii ; 11: R . typhi ; 12: E . chaffeensis ; 13: A . phagocytophilum ; 14: C . burnetii ; 15: E . coli ; 16: S . typhi .

    Techniques Used: Lamp Assay, Agarose Gel Electrophoresis, Positive Control

    12) Product Images from "Hierarchical control of enzymatic actuators using DNA-based switchable memories"

    Article Title: Hierarchical control of enzymatic actuators using DNA-based switchable memories

    Journal: Nature Communications

    doi: 10.1038/s41467-017-01127-w

    The control of enzymatic actuators by the translator module. a Schematic illustration (left) and experimental results (right) of controlling a NanoLuc (NL)-based actuator by the translator module. Experiments were performed using 2 nM of αtoσ , 5 nM of the NanoLuc-based actuator, 15 U mL −1 Bst 2.0 WarmStart DNA polymerase, 10 U mL −1 Nt. bstNBI, and initiated with 30 nM α . The opening of the stem-loop structure of the NanoLuc-based actuator was quantified at intervals of 30 min by measuring the BRET ratio between the NanoLuc donor (em. = 458 nm) and FAM acceptor dye (em. = 533 nm). The translator module was omitted for negative (−) and positive (+) controls and excess of DNA strand σ was added for the positive controls. Experiments were performed in triplicate and the fraction in opened conformation in normalized units (n.u.) was calculated by subtracting the mean BRET ratio of the positive controls and normalizing to the mean BRET ratio of the negative controls. Error bars and shaded areas represent the standard error of the mean of the experiments. b Schematic illustration (left) and experimental results (right) of the activation of the self-inhibitory TEM1 β -lactamase (β-lac) actuator by the translator module. Experiments were performed using 12 nM βtoξ , 2.5 nM TEM1 β -lactamase/BLIP actuator, 15 U mL −1 Bst 2.0 WarmStart DNA polymerase, 10 U mL −1 Nt. bstNBI, and initiated with 30 nM β . The activity of TEM1 β -lactamase was measured at time = 0 min prior to initiation with β and 30 min after activation of the translator module and was quantified by measuring the hydrolysis rate of fluorogenic substrate CCF2-FA obtained from the linear regime of the fluorescent time traces. The translator module was omitted for negative (−) and positive (+) controls and excess of DNA strand ξ was added for the positive controls. Experiments were performed in triplicate and the activity in normalized units (n.u.) was calculated by subtracting the mean hydrolysis rate of the negative controls and normalizing to the mean hydrolysis rate of the positive controls. Error bars and shaded area’s represent the standard error of the mean of the experiments
    Figure Legend Snippet: The control of enzymatic actuators by the translator module. a Schematic illustration (left) and experimental results (right) of controlling a NanoLuc (NL)-based actuator by the translator module. Experiments were performed using 2 nM of αtoσ , 5 nM of the NanoLuc-based actuator, 15 U mL −1 Bst 2.0 WarmStart DNA polymerase, 10 U mL −1 Nt. bstNBI, and initiated with 30 nM α . The opening of the stem-loop structure of the NanoLuc-based actuator was quantified at intervals of 30 min by measuring the BRET ratio between the NanoLuc donor (em. = 458 nm) and FAM acceptor dye (em. = 533 nm). The translator module was omitted for negative (−) and positive (+) controls and excess of DNA strand σ was added for the positive controls. Experiments were performed in triplicate and the fraction in opened conformation in normalized units (n.u.) was calculated by subtracting the mean BRET ratio of the positive controls and normalizing to the mean BRET ratio of the negative controls. Error bars and shaded areas represent the standard error of the mean of the experiments. b Schematic illustration (left) and experimental results (right) of the activation of the self-inhibitory TEM1 β -lactamase (β-lac) actuator by the translator module. Experiments were performed using 12 nM βtoξ , 2.5 nM TEM1 β -lactamase/BLIP actuator, 15 U mL −1 Bst 2.0 WarmStart DNA polymerase, 10 U mL −1 Nt. bstNBI, and initiated with 30 nM β . The activity of TEM1 β -lactamase was measured at time = 0 min prior to initiation with β and 30 min after activation of the translator module and was quantified by measuring the hydrolysis rate of fluorogenic substrate CCF2-FA obtained from the linear regime of the fluorescent time traces. The translator module was omitted for negative (−) and positive (+) controls and excess of DNA strand ξ was added for the positive controls. Experiments were performed in triplicate and the activity in normalized units (n.u.) was calculated by subtracting the mean hydrolysis rate of the negative controls and normalizing to the mean hydrolysis rate of the positive controls. Error bars and shaded area’s represent the standard error of the mean of the experiments

    Techniques Used: Bioluminescence Resonance Energy Transfer, Activation Assay, Activity Assay

    Control of two orthogonal enzymatic actuators by a switchable memories circuit. a Schematics of the experiment in which the switch controls a NanoLuc-based actuator and a self-inhibitory TEM1 β -lactamase construct. b – d Results of the experiments, carried out as described in the Methods, using 20 nM βtoiα , 15 nM αtoiβ , 24 nM βtoβ , 10 nM αtoα , γtoα , and δtoβ , 15 U mL −1 Bst 2.0 WarmStart DNA polymerase, 10 U mL −1 Nt. bstNBI, and 200 nM ttRecJ. Reactions were performed in presence of the actuators or MBs. The switch was initiated with 1 nM α . b The graphs show the dynamics of the switch and the production of σ and ξ measured using MBs (5 nM MB σ and 2.5 nM MB ξ ) (Supplementary Fig. 17 ) in absence (light color) and in presence (dark color) of the translator modules (2 nM αtoσ and 12 nM βtoξ ). The charge level is the normalized fluorescence of the signal of DY530 and FAM fluorophores, which is 0 in the absence of template’s input primer and 1 at the steady-state value of β and α , respectively. The dotted lines show the time points at which 30 nM of the Inputs δ and γ were added. In parallel, experiments were run where the MBs were replaced with the enzymatic actuators (5 nM of the NanoLuc-based actuator and 2.5 nM TEM1 β -lactamase actuator). c , d The state of the actuators was measured at four time points including negative (−) and positive (+) controls (Supplementary Figs. 13 , 14 and Methods). Error bars and shaded area’s represent the standard error of the mean of the experiments. Experiments were performed in plurality ( > 3) and at three different days. c The bar graphs displaying the BRET ratio were normalized to the mean of the negative controls for a clear visualization (Supplementary Fig. 14 displays the raw data). d The activity or fraction in opened conformation of the actuators were calculated by normalizing to positive and negative controls (Methods)
    Figure Legend Snippet: Control of two orthogonal enzymatic actuators by a switchable memories circuit. a Schematics of the experiment in which the switch controls a NanoLuc-based actuator and a self-inhibitory TEM1 β -lactamase construct. b – d Results of the experiments, carried out as described in the Methods, using 20 nM βtoiα , 15 nM αtoiβ , 24 nM βtoβ , 10 nM αtoα , γtoα , and δtoβ , 15 U mL −1 Bst 2.0 WarmStart DNA polymerase, 10 U mL −1 Nt. bstNBI, and 200 nM ttRecJ. Reactions were performed in presence of the actuators or MBs. The switch was initiated with 1 nM α . b The graphs show the dynamics of the switch and the production of σ and ξ measured using MBs (5 nM MB σ and 2.5 nM MB ξ ) (Supplementary Fig. 17 ) in absence (light color) and in presence (dark color) of the translator modules (2 nM αtoσ and 12 nM βtoξ ). The charge level is the normalized fluorescence of the signal of DY530 and FAM fluorophores, which is 0 in the absence of template’s input primer and 1 at the steady-state value of β and α , respectively. The dotted lines show the time points at which 30 nM of the Inputs δ and γ were added. In parallel, experiments were run where the MBs were replaced with the enzymatic actuators (5 nM of the NanoLuc-based actuator and 2.5 nM TEM1 β -lactamase actuator). c , d The state of the actuators was measured at four time points including negative (−) and positive (+) controls (Supplementary Figs. 13 , 14 and Methods). Error bars and shaded area’s represent the standard error of the mean of the experiments. Experiments were performed in plurality ( > 3) and at three different days. c The bar graphs displaying the BRET ratio were normalized to the mean of the negative controls for a clear visualization (Supplementary Fig. 14 displays the raw data). d The activity or fraction in opened conformation of the actuators were calculated by normalizing to positive and negative controls (Methods)

    Techniques Used: Construct, Fluorescence, Bioluminescence Resonance Energy Transfer, Activity Assay

    Characterizing retroactivity from coupling of the translator module to the memories circuit. a Schematic illustration of the system, in which the translator module is coupled to α or β of the PEN-based bistable switch. The core of the bistable switch consists of four templates, including the autocatalytic templates αtoα and βtoβ and the inhibitory templates αtoiβ and βtoiα . The network switches between states upon injection of γ and δ which are received by templates γtoα and δtoβ . The dynamics of the bistable switch are followed via N-quenching using templates βtoiα and αtoiβ which are 3′-end labeled with a DY530 and FAM fluorophore, respectively. b Experimental (Exp.) and simulated (Sim.) phase diagrams for a concentration range of translator template coupled to α or β . Experiments were carried out as described in the Methods using 20 nM βtoiα , 15 nM αtoiβ , 24 nM βtoβ , 10 nM αtoα , γtoα , and δtoβ , 15 U mL −1 Bst 2.0 WarmStart DNA polymerase, 10 U mL −1 Nt. bstNBI, and 200 nM ttRecJ. The switch was either equilibrated to its α-state and 30 nM δ was injected for switching to the β-state or the switch was equilibrated to its β-state and 30 nM γ was injected for switching to the α-state. The charge level is the normalized fluorescence of the signal of DY530 and FAM fluorophores, which is 0 in the absence of template’s input primer and 1 at the steady-state value of primer β and α , respectively. The blue and green circles represent the α-state and β-state, respectively. Simulations were performed using the heuristic model ( Supplementary Notes ) and the traces were converted to normalized units (n.u.) by normalizing α and β to their steady-state concentrations. c Bifurcation diagrams of the switch in isolation and with 10 nM of translator module coupled to β or α as a function of inputs γ and δ obtained using the heuristic model ( Supplementary Notes ). The monostable domains of α and β are shown in blue and green, respectively, while the bistable domain is shown in purple
    Figure Legend Snippet: Characterizing retroactivity from coupling of the translator module to the memories circuit. a Schematic illustration of the system, in which the translator module is coupled to α or β of the PEN-based bistable switch. The core of the bistable switch consists of four templates, including the autocatalytic templates αtoα and βtoβ and the inhibitory templates αtoiβ and βtoiα . The network switches between states upon injection of γ and δ which are received by templates γtoα and δtoβ . The dynamics of the bistable switch are followed via N-quenching using templates βtoiα and αtoiβ which are 3′-end labeled with a DY530 and FAM fluorophore, respectively. b Experimental (Exp.) and simulated (Sim.) phase diagrams for a concentration range of translator template coupled to α or β . Experiments were carried out as described in the Methods using 20 nM βtoiα , 15 nM αtoiβ , 24 nM βtoβ , 10 nM αtoα , γtoα , and δtoβ , 15 U mL −1 Bst 2.0 WarmStart DNA polymerase, 10 U mL −1 Nt. bstNBI, and 200 nM ttRecJ. The switch was either equilibrated to its α-state and 30 nM δ was injected for switching to the β-state or the switch was equilibrated to its β-state and 30 nM γ was injected for switching to the α-state. The charge level is the normalized fluorescence of the signal of DY530 and FAM fluorophores, which is 0 in the absence of template’s input primer and 1 at the steady-state value of primer β and α , respectively. The blue and green circles represent the α-state and β-state, respectively. Simulations were performed using the heuristic model ( Supplementary Notes ) and the traces were converted to normalized units (n.u.) by normalizing α and β to their steady-state concentrations. c Bifurcation diagrams of the switch in isolation and with 10 nM of translator module coupled to β or α as a function of inputs γ and δ obtained using the heuristic model ( Supplementary Notes ). The monostable domains of α and β are shown in blue and green, respectively, while the bistable domain is shown in purple

    Techniques Used: Injection, Labeling, Concentration Assay, Fluorescence, Isolation

    Coupling of the translator module to an upstream INVERTER network. a Schematic illustration of the translator module coupled to a PEN-based INVERTER network. Multiplex monitoring of the dynamics of the network is performed using endogenous template βtoiα and an exogenous template αtoiβ which are 3′-end fluorescently labeled with DY530 and FAM, respectively, while the output strand σ of the translator module is measured via a MB bearing a fluorophore-quencher pair. b Results of the experiments that were conducted for 0, 2, 5, 10, 20, and 40 nM (light to dark) of translator template αtoσ in the presence of 7 nM αtoα , 20 nM of βtoiα and αtoiβ , 30 nM MB, 10 U mL −1 Bst 2.0 WarmStart DNA polymerase, 25 U mL −1 Nt. bstNBI, and 50 nM ttRecJ. The INVERTER is activated by addition of 0.5 nM α which is initially amplified until it reaches steady-state in which production by polymerase and nickase and degradation due to exonuclease are balanced. Applying a pulse of 30 nM of input β at this point initiates the production of iα , which inhibits autocatalytic production of output α . As input strand β gets degraded the system returns its pre-stimulus steady-state. Hence, the INVERTER network shows a pulse response after injection of input β , which can be characterized by its amplitude and response time which is the time needed to recover to the pre-stimulus steady-state. The charge level is the normalized fluorescence of the signal of DY530 and FAM fluorophores, which is 0 in the absence of template’s input primer and 1 at the maximal or steady-state value of primer β and α , respectively. The fluorescence of Cy5 fluorophore was converted to concentration of DNA strand σ using a standard curve (Supplementary Fig. 17 ). c Results of simulations using the heuristic model with the same concentrations of translator template as used during the experiments in b and for different values of ρ . The traces were converted to normalized units (n.u.) by normalizing α to the steady-state concentration and normalizing β to its maximum value
    Figure Legend Snippet: Coupling of the translator module to an upstream INVERTER network. a Schematic illustration of the translator module coupled to a PEN-based INVERTER network. Multiplex monitoring of the dynamics of the network is performed using endogenous template βtoiα and an exogenous template αtoiβ which are 3′-end fluorescently labeled with DY530 and FAM, respectively, while the output strand σ of the translator module is measured via a MB bearing a fluorophore-quencher pair. b Results of the experiments that were conducted for 0, 2, 5, 10, 20, and 40 nM (light to dark) of translator template αtoσ in the presence of 7 nM αtoα , 20 nM of βtoiα and αtoiβ , 30 nM MB, 10 U mL −1 Bst 2.0 WarmStart DNA polymerase, 25 U mL −1 Nt. bstNBI, and 50 nM ttRecJ. The INVERTER is activated by addition of 0.5 nM α which is initially amplified until it reaches steady-state in which production by polymerase and nickase and degradation due to exonuclease are balanced. Applying a pulse of 30 nM of input β at this point initiates the production of iα , which inhibits autocatalytic production of output α . As input strand β gets degraded the system returns its pre-stimulus steady-state. Hence, the INVERTER network shows a pulse response after injection of input β , which can be characterized by its amplitude and response time which is the time needed to recover to the pre-stimulus steady-state. The charge level is the normalized fluorescence of the signal of DY530 and FAM fluorophores, which is 0 in the absence of template’s input primer and 1 at the maximal or steady-state value of primer β and α , respectively. The fluorescence of Cy5 fluorophore was converted to concentration of DNA strand σ using a standard curve (Supplementary Fig. 17 ). c Results of simulations using the heuristic model with the same concentrations of translator template as used during the experiments in b and for different values of ρ . The traces were converted to normalized units (n.u.) by normalizing α to the steady-state concentration and normalizing β to its maximum value

    Techniques Used: Multiplex Assay, Labeling, Amplification, Injection, Fluorescence, Concentration Assay

    13) Product Images from "Diagnosis of Brugian Filariasis by Loop-Mediated Isothermal Amplification"

    Article Title: Diagnosis of Brugian Filariasis by Loop-Mediated Isothermal Amplification

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0001948

    Species-specificity of Hha I LAMP assay. (A) Each curve represents the calculated average of triplicate turbidity curves generated with various genomic DNAs (0. 1 ng) using Bst 2.0 DNA polymerase without loop primers. Turbidity was observed using B. malayi or B. timori DNA. (B) As a positive control, an actin gene fragment was PCR amplified from B. malayi (Bma), D. immitis (Dim), O. volvulus (Ovo), the mosquito Aedes albopictus (Aal), W. bancrofti (Wba), human (Hsa) and B. timori (Bti) DNAs using degenerate primers. Agarose gel showing amplification of a 244 bp fragment of the actin gene. The 100 bp DNA Ladder (New England Biolabs) was used as the molecular weight marker (MWM). Water was used in the non-template controls (NTC) in (A) and (B).
    Figure Legend Snippet: Species-specificity of Hha I LAMP assay. (A) Each curve represents the calculated average of triplicate turbidity curves generated with various genomic DNAs (0. 1 ng) using Bst 2.0 DNA polymerase without loop primers. Turbidity was observed using B. malayi or B. timori DNA. (B) As a positive control, an actin gene fragment was PCR amplified from B. malayi (Bma), D. immitis (Dim), O. volvulus (Ovo), the mosquito Aedes albopictus (Aal), W. bancrofti (Wba), human (Hsa) and B. timori (Bti) DNAs using degenerate primers. Agarose gel showing amplification of a 244 bp fragment of the actin gene. The 100 bp DNA Ladder (New England Biolabs) was used as the molecular weight marker (MWM). Water was used in the non-template controls (NTC) in (A) and (B).

    Techniques Used: Lamp Assay, Generated, Positive Control, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Molecular Weight, Marker

    Sensitivity of Hha I LAMP assay. Ten-fold serial dilutions of B. malayi genomic DNA amplified with the Hha I primer set alone (A) or in the presence of loop primers (B) with Bst DNA polymerase, large fragment (wt Bst LF), Bst 2.0 DNA polymerase ( Bst 2.0) and Bst 2.0 WarmStart DNA polymerase ( Bst 2.0 WS). Data points represent the average of three samples and the error bars represent the standard deviation at each point. For each enzyme, the average threshold time, defined as the time at which the change in turbidity over time (dT/dt) reaches a value of 0.1, is plotted against the amount of starting material. (C) UV detection (365 nm) of products generated within 60 minutes using Bst 2.0 in the presence of loop primers and Fluorescent Detection Reagent. The amount of starting material in ng is shown below the photograph. Positive samples fluoresce green while negative samples remain dark.
    Figure Legend Snippet: Sensitivity of Hha I LAMP assay. Ten-fold serial dilutions of B. malayi genomic DNA amplified with the Hha I primer set alone (A) or in the presence of loop primers (B) with Bst DNA polymerase, large fragment (wt Bst LF), Bst 2.0 DNA polymerase ( Bst 2.0) and Bst 2.0 WarmStart DNA polymerase ( Bst 2.0 WS). Data points represent the average of three samples and the error bars represent the standard deviation at each point. For each enzyme, the average threshold time, defined as the time at which the change in turbidity over time (dT/dt) reaches a value of 0.1, is plotted against the amount of starting material. (C) UV detection (365 nm) of products generated within 60 minutes using Bst 2.0 in the presence of loop primers and Fluorescent Detection Reagent. The amount of starting material in ng is shown below the photograph. Positive samples fluoresce green while negative samples remain dark.

    Techniques Used: Lamp Assay, Amplification, Standard Deviation, Generated

    Hha I LAMP assay for the detection of B. malayi infected blood samples. A set of serial dilutions (two-fold) of microfilariae in blood was prepared and DNA was isolated from each dilution. Three experiments were performed using a different but overlapping range of DNA dilutions. One µl of DNA from each dilution was used in LAMP reactions with Bst 2.0 DNA polymerase. Samples from each experimental set-up were performed in triplicate (experiments 1 and 2) or duplicate (experiment 3). Average threshold times and standard deviations were plotted against the approximate number of mf/µl DNA solution.
    Figure Legend Snippet: Hha I LAMP assay for the detection of B. malayi infected blood samples. A set of serial dilutions (two-fold) of microfilariae in blood was prepared and DNA was isolated from each dilution. Three experiments were performed using a different but overlapping range of DNA dilutions. One µl of DNA from each dilution was used in LAMP reactions with Bst 2.0 DNA polymerase. Samples from each experimental set-up were performed in triplicate (experiments 1 and 2) or duplicate (experiment 3). Average threshold times and standard deviations were plotted against the approximate number of mf/µl DNA solution.

    Techniques Used: Lamp Assay, Infection, Isolation

    14) Product Images from "Development of a loop-mediated isothermal amplification (LAMP) assay for rapid screening of ticks and fleas for spotted fever group rickettsia"

    Article Title: Development of a loop-mediated isothermal amplification (LAMP) assay for rapid screening of ticks and fleas for spotted fever group rickettsia

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0192331

    Specificity of SFGR-LAMP assay using Bst 2.0 WarmStart DNA polymerase. (A). Colorimetric visual detection of hydroxynaphol blue-based SFGR-LAMP reaction products inspected by the naked eye. The color changes from light blue in the positive reactions to dark blue to purple in the negative reactions. (B). Agarose gel electrophoresis. Lane 1: 100Kbp molecular ladder; 2: water control; 3: positive control ( R . rickettsii ); 4: R . africae ; 5: R . amblyommatis ; 6: R . conorii ; 7: R . felis ; 8: R . montanensis ; 9: R . parkeri ; 10: R . rickettsii ; 11: R . typhi ; 12: E . chaffeensis ; 13: A . phagocytophilum ; 14: C . burnetii ; 15: E . coli ; 16: S . typhi .
    Figure Legend Snippet: Specificity of SFGR-LAMP assay using Bst 2.0 WarmStart DNA polymerase. (A). Colorimetric visual detection of hydroxynaphol blue-based SFGR-LAMP reaction products inspected by the naked eye. The color changes from light blue in the positive reactions to dark blue to purple in the negative reactions. (B). Agarose gel electrophoresis. Lane 1: 100Kbp molecular ladder; 2: water control; 3: positive control ( R . rickettsii ); 4: R . africae ; 5: R . amblyommatis ; 6: R . conorii ; 7: R . felis ; 8: R . montanensis ; 9: R . parkeri ; 10: R . rickettsii ; 11: R . typhi ; 12: E . chaffeensis ; 13: A . phagocytophilum ; 14: C . burnetii ; 15: E . coli ; 16: S . typhi .

    Techniques Used: Lamp Assay, Agarose Gel Electrophoresis, Positive Control

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    Real-time Polymerase Chain Reaction:

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    Incubation:

    Article Title: Massively parallel biophysical analysis of CRISPR-Cas complexes on next generation sequencing chips
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    Modification:

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    Hybridization:

    Article Title: Massively parallel biophysical analysis of CRISPR-Cas complexes on next generation sequencing chips
    Article Snippet: After denaturation, the chip was heated to 85°C and incubated with 500 nM of the regeneration primer (CJ.RP) in hybridization buffer (75 mM Trisodium Citrate, pH 7.0, 750 mM NaCl, 0.1% Tween-20). .. CJ.RP was extended at 60°C for 10 minutes in isothermal amplification buffer (20 mM Tris-HCl, pH 8.8, 10 mM (NH4 )2 SO4 , 50 mM KCl, 2 mM MgSO4 , 0.1% Tween-20) containing 0.08 U/l of Bst 2.0 WarmStart DNA polymerase (New England Biolabs) and 0.8 mM of dNTPs.

    Serial Dilution:

    Article Title: Two Methods for Increased Specificity and Sensitivity in Loop-Mediated Isothermal Amplification
    Article Snippet: The optimized LAMP mixture was used with the Touchdown methodology to detect a serial dilution of L. monocytogenes DNA template. .. After the mixtures were preheated at 95 °C for 5 min and Bst 2.0 WarmStart DNA polymerase (New England Biolabs, Beverly, MA, USA) were added, they were heated at 63 °C for 5 min, at 61 °C for 5 min, at 59 °C for 5 min and then at 57 °C for 60 min, and, as indicated in , the sensitivity of Touchdown LAMP was found to be 10 fg of L. monocytogenes DNA.

    Generated:

    Article Title: Evaluation of an Internally Controlled Multiplex Tth Endonuclease Cleavage Loop-Mediated Isothermal Amplification (TEC-LAMP) Assay for the Detection of Bacterial Meningitis Pathogens
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    other:

    Article Title: Diagnosis of Brugian Filariasis by Loop-Mediated Isothermal Amplification
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    Sequencing:

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    Article Snippet: The SmMIT-LAMP method amplifies a specific sequence corresponding to a mitochondrial S . mansoni minisatellite DNA region (GenBank Acc. .. Briefly, the reaction was carried out in 25 μL reaction mixture containing 40 pmol each of FIP and BIP primers, 5 pmol of each F3 and B3 primers, 1.4 mM of each dNTP (Intron), 1x Isothermal Amplification Buffer -20 mM Tris-HCl (pH 8.8), 50 mM KCl, 10 mM (NH4 )2 SO4 , 2 mM MgSO4 , 0.1% Tween20- (New England Biolabs, UK), 1 M betaine (Sigma, USA), supplementary 6 mM of MgSO4 (New England Biolabs, UK) and 8 U of Bst 2.0 WarmStart DNA polymerase (New England Biolabs, UK) with 2 μL of template DNA.

    Article Title: Massively parallel biophysical analysis of CRISPR-Cas complexes on next generation sequencing chips
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    Article Title: DNA methylation variation of human-specific Alu repeats
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    Combined Bisulfite Restriction Analysis Assay:

    Article Title: DNA methylation variation of human-specific Alu repeats
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    Fluorescence:

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    Labeling:

    Article Title: Massively parallel biophysical analysis of CRISPR-Cas complexes on next generation sequencing chips
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    Purification:

    Article Title: DNA methylation variation of human-specific Alu repeats
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    Article Title: Development of a loop-mediated isothermal amplification (LAMP) assay for rapid screening of ticks and fleas for spotted fever group rickettsia
    Article Snippet: Two commercially available LAMP chemistries were used: the Bst 2.0 WarmStart DNA Polymerase (New England Biolabs, USA) and the GspSSD2.0 Isothermal Master Mix (ISO-004) (Optigene, UK). .. Amplicons from F3/B3 LAMP primers were purified using a PCR purification kit (Qiagen) and sequenced in the Oklahoma State University core facility to confirm that the targeted region amplified by the LAMP assay matched with the expected 17kDa protein gene of R . rickettsii .

    Polymerase Chain Reaction:

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    Article Title: DNA methylation variation of human-specific Alu repeats
    Article Snippet: Multiple PCR products of the same sample were combined into one barcoded library, with different barcodes for the different samples. .. These libraries were then end-repaired and ligated to Ion Torrent “A” and “P1” adaptors lacking 5′-phosphate, using a novel scheme that uses T4 ligase and Bst 2.0 WarmStart® DNA Polymerase (New England BioLabs).

    Article Title: Development of a loop-mediated isothermal amplification (LAMP) assay for rapid screening of ticks and fleas for spotted fever group rickettsia
    Article Snippet: Two commercially available LAMP chemistries were used: the Bst 2.0 WarmStart DNA Polymerase (New England Biolabs, USA) and the GspSSD2.0 Isothermal Master Mix (ISO-004) (Optigene, UK). .. Amplicons from F3/B3 LAMP primers were purified using a PCR purification kit (Qiagen) and sequenced in the Oklahoma State University core facility to confirm that the targeted region amplified by the LAMP assay matched with the expected 17kDa protein gene of R . rickettsii .

    IA:

    Article Title: First Characterization of a Biphasic, Switch-like DNA Amplification
    Article Snippet: Nuclease-free water and oligo length standard 10/60 were purchased from Integrated DNA Technologies, Inc. (Coralville, IA). .. Nt.BstNBI nicking endonuclease, Bst 2.0 WarmStart® DNA Polymerase, 10× ThermoPol I Buffer, dNTPs, BSA, and 100 mM MgSO4 were purchased from New England Biolabs (Beverly, MA).

    Chromatin Immunoprecipitation:

    Article Title: Massively parallel biophysical analysis of CRISPR-Cas complexes on next generation sequencing chips
    Article Snippet: Paragraph title: Chip regeneration and addition of alignment markers ... CJ.RP was extended at 60°C for 10 minutes in isothermal amplification buffer (20 mM Tris-HCl, pH 8.8, 10 mM (NH4 )2 SO4 , 50 mM KCl, 2 mM MgSO4 , 0.1% Tween-20) containing 0.08 U/l of Bst 2.0 WarmStart DNA polymerase (New England Biolabs) and 0.8 mM of dNTPs.

    Article Title: DNA methylation variation of human-specific Alu repeats
    Article Snippet: These libraries were then end-repaired and ligated to Ion Torrent “A” and “P1” adaptors lacking 5′-phosphate, using a novel scheme that uses T4 ligase and Bst 2.0 WarmStart® DNA Polymerase (New England BioLabs). .. Next, each library, with its unique barcode, was combined for multiplexed next-generation-sequencing using the Ion Torrent 318-v2 chip as described above for HT-TREBS.

    SYBR Green Assay:

    Article Title: A field survey using LAMP assay for detection of Schistosoma mansoni in a low-transmission area of schistosomiasis in Umbuzeiro, Brazil: Assessment in human and snail samples
    Article Snippet: Briefly, the reaction was carried out in 25 μL reaction mixture containing 40 pmol each of FIP and BIP primers, 5 pmol of each F3 and B3 primers, 1.4 mM of each dNTP (Intron), 1x Isothermal Amplification Buffer -20 mM Tris-HCl (pH 8.8), 50 mM KCl, 10 mM (NH4 )2 SO4 , 2 mM MgSO4 , 0.1% Tween20- (New England Biolabs, UK), 1 M betaine (Sigma, USA), supplementary 6 mM of MgSO4 (New England Biolabs, UK) and 8 U of Bst 2.0 WarmStart DNA polymerase (New England Biolabs, UK) with 2 μL of template DNA. .. The LAMP-positive results could be visually inspected by naked eye by color change after adding 2 μL of 1:10 diluted 10,000x concentration fluorescent dye SYBR Green I to the reactions tubes.

    Article Title: First Characterization of a Biphasic, Switch-like DNA Amplification
    Article Snippet: UltraPure™ Tris-HCI pH 8.0, RNase free EDTA, RNase free MgCl2 , RNase free KCl, Novex™ TBE Running Buffer (5X), 2X TBE-Urea Sample Buffer, Novex™ TBE-Urea Gels, 15%, SYBR® Gold Nucleic Acid Gel Stain, and SYBR® Green II RNA Gel Stain were purchased from Thermo Fisher Scientific (Waltham, MA). .. Nt.BstNBI nicking endonuclease, Bst 2.0 WarmStart® DNA Polymerase, 10× ThermoPol I Buffer, dNTPs, BSA, and 100 mM MgSO4 were purchased from New England Biolabs (Beverly, MA).

    Multiplex Assay:

    Article Title: Evaluation of an Internally Controlled Multiplex Tth Endonuclease Cleavage Loop-Mediated Isothermal Amplification (TEC-LAMP) Assay for the Detection of Bacterial Meningitis Pathogens
    Article Snippet: Paragraph title: 4.3. Internally Controlled Multiplex TEC-LAMP Assay ... The final TEC-LAMP reaction contained 1× Isothermal Amplification Buffer (New England Biolabs, Hitchin, UK), 6 mM MgSO4 (Roche Diagnostics), 1.4 mM deoxynucleotide triphosphate set (New England Biolabs), S. pneumoniae oligonucleotides [2.6 µM reverse inner, 1.3 µM forward inner and TEC primer/probe, 0.65 µM forward and reverse loop, 0.325 µM forward and reverse outer], N. meningitis oligonucleotides [1 µM reverse inner, 0.5 µM forward inner and TEC primer/probe, 0.25 µM forward and reverse loop, 0.125 µM forward and reverse outer], H. influenzae oligonucleotides [1.2 µM reverse inner, 0.6 µM forward inner and TEC primer/probe, 0.3 µM forward and reverse loop, 0.15 µM forward and reverse outer], IAC oligonucleotides [0.6 µM reverse inner, 0.3 µM forward inner and TEC primer/probe, 0.15 µM forward and reverse loop, 0.075 µM forward and reverse outer], 8 U Bst 2.0 WarmStart DNA polymerase (New England Biolabs), 15 U Tth endonuclease IV (New England Biolabs), 1 µL IAC template (50 copies), 1 µL DNA template (1–3 templates) or 1 µL molecular grade water for no template control (NTC) reactions, and molecular grade water to give a final volume of 25 µL.

    Agarose Gel Electrophoresis:

    Article Title: Development of loop-mediated isothermal amplification (LAMP) for simple detection of Leishmania infection
    Article Snippet: Loop-mediated isothermal amplification The LAMP reaction mixtures (25 μl) were based on that described by Tomita et al. [ ], which contained 1× Isothermal Amplification Buffer (New England Biolabs, USA), 8 mM MgSO4 , 0.8 M Betaine (Sigma-Aldrich, USA), 1.4 mM each of dATP, dCTP, dGTP, and dTTP (SibEnzyme, Russia), 40 pmol of FIP primer, 40 pmol of BIP primer, 10 pmol of F3 primer, 10 pmol of B3 primer, and 8 U of Bst 2.0 WarmStart® DNA Polymerase (New England Biolabs, USA). .. Visualization of the LAMP products was performed using 2.5 % agarose gel electrophoresis at 10 V/cm in 1× TAE buffer.

    Article Title: DNA methylation variation of human-specific Alu repeats
    Article Snippet: These libraries were then end-repaired and ligated to Ion Torrent “A” and “P1” adaptors lacking 5′-phosphate, using a novel scheme that uses T4 ligase and Bst 2.0 WarmStart® DNA Polymerase (New England BioLabs). .. Unligated adaptors were then removed by agarose gel extraction and further purification by Select-A-Size DNA columns (Zymo Research), and the libraries were quantified on the Bioanalyzer (Agilent Technologies).

    Next-Generation Sequencing:

    Article Title: DNA methylation variation of human-specific Alu repeats
    Article Snippet: One set of PCR products used for the COBRA analyses was used for NGS-based bisulfite sequencing. .. These libraries were then end-repaired and ligated to Ion Torrent “A” and “P1” adaptors lacking 5′-phosphate, using a novel scheme that uses T4 ligase and Bst 2.0 WarmStart® DNA Polymerase (New England BioLabs).

    Concentration Assay:

    Article Title: A field survey using LAMP assay for detection of Schistosoma mansoni in a low-transmission area of schistosomiasis in Umbuzeiro, Brazil: Assessment in human and snail samples
    Article Snippet: Briefly, the reaction was carried out in 25 μL reaction mixture containing 40 pmol each of FIP and BIP primers, 5 pmol of each F3 and B3 primers, 1.4 mM of each dNTP (Intron), 1x Isothermal Amplification Buffer -20 mM Tris-HCl (pH 8.8), 50 mM KCl, 10 mM (NH4 )2 SO4 , 2 mM MgSO4 , 0.1% Tween20- (New England Biolabs, UK), 1 M betaine (Sigma, USA), supplementary 6 mM of MgSO4 (New England Biolabs, UK) and 8 U of Bst 2.0 WarmStart DNA polymerase (New England Biolabs, UK) with 2 μL of template DNA. .. The LAMP-positive results could be visually inspected by naked eye by color change after adding 2 μL of 1:10 diluted 10,000x concentration fluorescent dye SYBR Green I to the reactions tubes.

    Article Title: First Characterization of a Biphasic, Switch-like DNA Amplification
    Article Snippet: Nt.BstNBI nicking endonuclease, Bst 2.0 WarmStart® DNA Polymerase, 10× ThermoPol I Buffer, dNTPs, BSA, and 100 mM MgSO4 were purchased from New England Biolabs (Beverly, MA). .. Desalted amplification templates were purchased from Integrated DNA Technologies (Coralville, IA) suspended in IDTE Buffer at a concentration of 100 µM.

    Lamp Assay:

    Article Title: The Rapid-Heat LAMPellet Method: A Potential Diagnostic Method for Human Urogenital Schistosomiasis
    Article Snippet: .. Setting up LAMP assay To establish a standard procedure for the LAMP assay we used the Bst 2.0 WarmStart DNA polymerase applying a range of temperatures (61, 63 and 65°C) for testing different mixtures containing variable concentrations of betaine (ranging 0.8, 1 or 1.2 M) combined with supplementary variable concentrations of MgSO4 (ranging 4, 6 or 8 mM) in a heating block for 30, 50 and 60 min. .. The best amplification results were obtained when the reaction mixture contained 1 M of betaine combined with supplementary 6 mM of MgSO4 (resulting a final concentration of 8 mM MgSO4 in 1x Isothermal Amplification Buffer) and was incubated for 50 min at 63°C in a heating block ( ).

    Article Title: Development of a loop-mediated isothermal amplification (LAMP) assay for rapid screening of ticks and fleas for spotted fever group rickettsia
    Article Snippet: Six primers were designed for LAMP assay ( ) and were designed based on the 17kDa protein gene, the same region that has been used for end-point and qPCR primers to screen SFG rickettsia species in field-collected ticks [ , ]. .. Two commercially available LAMP chemistries were used: the Bst 2.0 WarmStart DNA Polymerase (New England Biolabs, USA) and the GspSSD2.0 Isothermal Master Mix (ISO-004) (Optigene, UK).

    Staining:

    Article Title: First Characterization of a Biphasic, Switch-like DNA Amplification
    Article Snippet: UltraPure™ Tris-HCI pH 8.0, RNase free EDTA, RNase free MgCl2 , RNase free KCl, Novex™ TBE Running Buffer (5X), 2X TBE-Urea Sample Buffer, Novex™ TBE-Urea Gels, 15%, SYBR® Gold Nucleic Acid Gel Stain, and SYBR® Green II RNA Gel Stain were purchased from Thermo Fisher Scientific (Waltham, MA). .. Nt.BstNBI nicking endonuclease, Bst 2.0 WarmStart® DNA Polymerase, 10× ThermoPol I Buffer, dNTPs, BSA, and 100 mM MgSO4 were purchased from New England Biolabs (Beverly, MA).

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    New England Biolabs bst2 0 warmstart polymerase
    Overall scheme for NGS-based deep bisulfite sequencing. (A) The entire procedure of the NGS-based deep bisulfite sequencing protocol is shown as a flow chart. (B) The adaptor ligation step for the current protocol has adopted one strategy, in which the added PCR products and two adaptors are ligated through two stepwise incubations. Since both adaptors lack the phosphate group at their 5′-ends, the ligation reaction by T4 at 25 °C occurs between only one strand of the adaptors and the PCR products. In this case, the phosphate groups are derived from the 5′-end of the PCR products. At 65 °C, the activated <t>Bst2.0</t> <t>WarmStart</t> polymerase extends and displaces the other unligated strand from the partially joined products. The * symbol indicates the phosphate group at the 5′-end of the end-repaired PCR products. (C) The sequences of Ion Torrent P1 and A adaptors are shown with different colors to indicate the key, barcode and spacer regions. The * symbol indicates a phosphothiate bonding between two nucleotides, which protects the duplex adaptor from being digested by the exonuclease activity of DNA polymerases.
    Bst2 0 Warmstart Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 45 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Overall scheme for NGS-based deep bisulfite sequencing. (A) The entire procedure of the NGS-based deep bisulfite sequencing protocol is shown as a flow chart. (B) The adaptor ligation step for the current protocol has adopted one strategy, in which the added PCR products and two adaptors are ligated through two stepwise incubations. Since both adaptors lack the phosphate group at their 5′-ends, the ligation reaction by T4 at 25 °C occurs between only one strand of the adaptors and the PCR products. In this case, the phosphate groups are derived from the 5′-end of the PCR products. At 65 °C, the activated Bst2.0 WarmStart polymerase extends and displaces the other unligated strand from the partially joined products. The * symbol indicates the phosphate group at the 5′-end of the end-repaired PCR products. (C) The sequences of Ion Torrent P1 and A adaptors are shown with different colors to indicate the key, barcode and spacer regions. The * symbol indicates a phosphothiate bonding between two nucleotides, which protects the duplex adaptor from being digested by the exonuclease activity of DNA polymerases.

    Journal: MethodsX

    Article Title: NGS-based deep bisulfite sequencing

    doi: 10.1016/j.mex.2015.11.008

    Figure Lengend Snippet: Overall scheme for NGS-based deep bisulfite sequencing. (A) The entire procedure of the NGS-based deep bisulfite sequencing protocol is shown as a flow chart. (B) The adaptor ligation step for the current protocol has adopted one strategy, in which the added PCR products and two adaptors are ligated through two stepwise incubations. Since both adaptors lack the phosphate group at their 5′-ends, the ligation reaction by T4 at 25 °C occurs between only one strand of the adaptors and the PCR products. In this case, the phosphate groups are derived from the 5′-end of the PCR products. At 65 °C, the activated Bst2.0 WarmStart polymerase extends and displaces the other unligated strand from the partially joined products. The * symbol indicates the phosphate group at the 5′-end of the end-repaired PCR products. (C) The sequences of Ion Torrent P1 and A adaptors are shown with different colors to indicate the key, barcode and spacer regions. The * symbol indicates a phosphothiate bonding between two nucleotides, which protects the duplex adaptor from being digested by the exonuclease activity of DNA polymerases.

    Article Snippet: The current protocol also uses a mixture of T4 ligase and Bst2.0 WarmStart polymerase with two stepwise incubations, a 25-min incubation at 25 °C for the ligation reaction by T4 ligase and another 30-min incubation at 65 °C for the extension/displacement reaction by Bst2.0 WarmStart polymerase (available from NEB).

    Techniques: Next-Generation Sequencing, Methylation Sequencing, Flow Cytometry, Ligation, Polymerase Chain Reaction, Derivative Assay, Activity Assay

    Species-specificity of Hha I LAMP assay. (A) Each curve represents the calculated average of triplicate turbidity curves generated with various genomic DNAs (0. 1 ng) using Bst 2.0 DNA polymerase without loop primers. Turbidity was observed using B. malayi or B. timori DNA. (B) As a positive control, an actin gene fragment was PCR amplified from B. malayi (Bma), D. immitis (Dim), O. volvulus (Ovo), the mosquito Aedes albopictus (Aal), W. bancrofti (Wba), human (Hsa) and B. timori (Bti) DNAs using degenerate primers. Agarose gel showing amplification of a 244 bp fragment of the actin gene. The 100 bp DNA Ladder (New England Biolabs) was used as the molecular weight marker (MWM). Water was used in the non-template controls (NTC) in (A) and (B).

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Diagnosis of Brugian Filariasis by Loop-Mediated Isothermal Amplification

    doi: 10.1371/journal.pntd.0001948

    Figure Lengend Snippet: Species-specificity of Hha I LAMP assay. (A) Each curve represents the calculated average of triplicate turbidity curves generated with various genomic DNAs (0. 1 ng) using Bst 2.0 DNA polymerase without loop primers. Turbidity was observed using B. malayi or B. timori DNA. (B) As a positive control, an actin gene fragment was PCR amplified from B. malayi (Bma), D. immitis (Dim), O. volvulus (Ovo), the mosquito Aedes albopictus (Aal), W. bancrofti (Wba), human (Hsa) and B. timori (Bti) DNAs using degenerate primers. Agarose gel showing amplification of a 244 bp fragment of the actin gene. The 100 bp DNA Ladder (New England Biolabs) was used as the molecular weight marker (MWM). Water was used in the non-template controls (NTC) in (A) and (B).

    Article Snippet: Reaction times were slightly slower using Bst 2.0 WarmStart DNA polymerase regardless of the presence of loop primers ( ).

    Techniques: Lamp Assay, Generated, Positive Control, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Molecular Weight, Marker

    Sensitivity of Hha I LAMP assay. Ten-fold serial dilutions of B. malayi genomic DNA amplified with the Hha I primer set alone (A) or in the presence of loop primers (B) with Bst DNA polymerase, large fragment (wt Bst LF), Bst 2.0 DNA polymerase ( Bst 2.0) and Bst 2.0 WarmStart DNA polymerase ( Bst 2.0 WS). Data points represent the average of three samples and the error bars represent the standard deviation at each point. For each enzyme, the average threshold time, defined as the time at which the change in turbidity over time (dT/dt) reaches a value of 0.1, is plotted against the amount of starting material. (C) UV detection (365 nm) of products generated within 60 minutes using Bst 2.0 in the presence of loop primers and Fluorescent Detection Reagent. The amount of starting material in ng is shown below the photograph. Positive samples fluoresce green while negative samples remain dark.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Diagnosis of Brugian Filariasis by Loop-Mediated Isothermal Amplification

    doi: 10.1371/journal.pntd.0001948

    Figure Lengend Snippet: Sensitivity of Hha I LAMP assay. Ten-fold serial dilutions of B. malayi genomic DNA amplified with the Hha I primer set alone (A) or in the presence of loop primers (B) with Bst DNA polymerase, large fragment (wt Bst LF), Bst 2.0 DNA polymerase ( Bst 2.0) and Bst 2.0 WarmStart DNA polymerase ( Bst 2.0 WS). Data points represent the average of three samples and the error bars represent the standard deviation at each point. For each enzyme, the average threshold time, defined as the time at which the change in turbidity over time (dT/dt) reaches a value of 0.1, is plotted against the amount of starting material. (C) UV detection (365 nm) of products generated within 60 minutes using Bst 2.0 in the presence of loop primers and Fluorescent Detection Reagent. The amount of starting material in ng is shown below the photograph. Positive samples fluoresce green while negative samples remain dark.

    Article Snippet: Reaction times were slightly slower using Bst 2.0 WarmStart DNA polymerase regardless of the presence of loop primers ( ).

    Techniques: Lamp Assay, Amplification, Standard Deviation, Generated

    Hha I LAMP assay for the detection of B. malayi infected blood samples. A set of serial dilutions (two-fold) of microfilariae in blood was prepared and DNA was isolated from each dilution. Three experiments were performed using a different but overlapping range of DNA dilutions. One µl of DNA from each dilution was used in LAMP reactions with Bst 2.0 DNA polymerase. Samples from each experimental set-up were performed in triplicate (experiments 1 and 2) or duplicate (experiment 3). Average threshold times and standard deviations were plotted against the approximate number of mf/µl DNA solution.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Diagnosis of Brugian Filariasis by Loop-Mediated Isothermal Amplification

    doi: 10.1371/journal.pntd.0001948

    Figure Lengend Snippet: Hha I LAMP assay for the detection of B. malayi infected blood samples. A set of serial dilutions (two-fold) of microfilariae in blood was prepared and DNA was isolated from each dilution. Three experiments were performed using a different but overlapping range of DNA dilutions. One µl of DNA from each dilution was used in LAMP reactions with Bst 2.0 DNA polymerase. Samples from each experimental set-up were performed in triplicate (experiments 1 and 2) or duplicate (experiment 3). Average threshold times and standard deviations were plotted against the approximate number of mf/µl DNA solution.

    Article Snippet: Reaction times were slightly slower using Bst 2.0 WarmStart DNA polymerase regardless of the presence of loop primers ( ).

    Techniques: Lamp Assay, Infection, Isolation

    Single-nucleotide polymorphism loop mediated isothermal amplification (SNP-LAMP) of mutant Y580 kelch13 from laboratory strain MRA 1240 (Y580, gray bars) versus wild-type MRA 1236 (C580, black bars). The SNP-LAMP was performed using Bst 2.0 WarmStart DNA polymerase at 63 o C with primer set 31. Turbidity was measured at 600-nm wavelength by NanoDrop at different reaction time points: (a) 50 minutes, (b) 55 minutes, (c) 60 minutes, (d) 65 minutes, (e) 70 minutes, and (f) 75 minutes.

    Journal: Open Forum Infectious Diseases

    Article Title: A Novel Single-Nucleotide Polymorphism Loop Mediated Isothermal Amplification Assay for Detection of Artemisinin-Resistant Plasmodium falciparum Malaria

    doi: 10.1093/ofid/ofy011

    Figure Lengend Snippet: Single-nucleotide polymorphism loop mediated isothermal amplification (SNP-LAMP) of mutant Y580 kelch13 from laboratory strain MRA 1240 (Y580, gray bars) versus wild-type MRA 1236 (C580, black bars). The SNP-LAMP was performed using Bst 2.0 WarmStart DNA polymerase at 63 o C with primer set 31. Turbidity was measured at 600-nm wavelength by NanoDrop at different reaction time points: (a) 50 minutes, (b) 55 minutes, (c) 60 minutes, (d) 65 minutes, (e) 70 minutes, and (f) 75 minutes.

    Article Snippet: LAMP conditions were optimized as follows: 20 mM Tris-HCl, 10 mM (NH4 )2 SO4 , 50 mM KCl, and 0.1% Tween 20 pH 8.8 (Isothermal Buffer, New England Biolabs, Whitby, ON), 8 mM MgSO4 , 0.8 M Betaine, and 8 units of Bst 2.0 WarmStart DNA polymerase (New England Biolabs, Whitby, ON).

    Techniques: Amplification, Mutagenesis

    Single-nucleotide polymorphism loop mediated isothermal amplification of mutant Y580 kelch13 from laboratory strain MRA 1240 (Y580, gray bars) versus wild-type MRA 1236 (C580, black bars) using Bst 2.0 WarmStart DNA polymerase at (a) 59°C, (b) 61°C, (c) 63°C, and (d) 65°C for 60 minutes. The template used was a polymerase chain reaction product of the kelch13 propellor domain with primer set 31.

    Journal: Open Forum Infectious Diseases

    Article Title: A Novel Single-Nucleotide Polymorphism Loop Mediated Isothermal Amplification Assay for Detection of Artemisinin-Resistant Plasmodium falciparum Malaria

    doi: 10.1093/ofid/ofy011

    Figure Lengend Snippet: Single-nucleotide polymorphism loop mediated isothermal amplification of mutant Y580 kelch13 from laboratory strain MRA 1240 (Y580, gray bars) versus wild-type MRA 1236 (C580, black bars) using Bst 2.0 WarmStart DNA polymerase at (a) 59°C, (b) 61°C, (c) 63°C, and (d) 65°C for 60 minutes. The template used was a polymerase chain reaction product of the kelch13 propellor domain with primer set 31.

    Article Snippet: LAMP conditions were optimized as follows: 20 mM Tris-HCl, 10 mM (NH4 )2 SO4 , 50 mM KCl, and 0.1% Tween 20 pH 8.8 (Isothermal Buffer, New England Biolabs, Whitby, ON), 8 mM MgSO4 , 0.8 M Betaine, and 8 units of Bst 2.0 WarmStart DNA polymerase (New England Biolabs, Whitby, ON).

    Techniques: Amplification, Mutagenesis, Polymerase Chain Reaction

    Amplification time (minutes) required for a positive call by 3 different enzymes using single-nucleotide polymorphism loop mediated isothermal amplification for Plasmodium falciparum -culture spiked whole blood: (a) GspSSD2 enzyme (63 o C); (b) Bst 2.0 WarmStart DNA polymerase (63 o C). These studies were performed on the CFX-96 detection system.

    Journal: Open Forum Infectious Diseases

    Article Title: A Novel Single-Nucleotide Polymorphism Loop Mediated Isothermal Amplification Assay for Detection of Artemisinin-Resistant Plasmodium falciparum Malaria

    doi: 10.1093/ofid/ofy011

    Figure Lengend Snippet: Amplification time (minutes) required for a positive call by 3 different enzymes using single-nucleotide polymorphism loop mediated isothermal amplification for Plasmodium falciparum -culture spiked whole blood: (a) GspSSD2 enzyme (63 o C); (b) Bst 2.0 WarmStart DNA polymerase (63 o C). These studies were performed on the CFX-96 detection system.

    Article Snippet: LAMP conditions were optimized as follows: 20 mM Tris-HCl, 10 mM (NH4 )2 SO4 , 50 mM KCl, and 0.1% Tween 20 pH 8.8 (Isothermal Buffer, New England Biolabs, Whitby, ON), 8 mM MgSO4 , 0.8 M Betaine, and 8 units of Bst 2.0 WarmStart DNA polymerase (New England Biolabs, Whitby, ON).

    Techniques: Amplification