bst large fragment polymerase  (New England Biolabs)


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    Name:
    Bst DNA Polymerase Lg Frag
    Description:
    Bst DNA Polymerase Lg Frag 8 000 units
    Catalog Number:
    m0275l
    Price:
    283
    Size:
    8 000 units
    Category:
    Thermostable DNA Polymerases
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    New England Biolabs bst large fragment polymerase
    Bst DNA Polymerase Lg Frag
    Bst DNA Polymerase Lg Frag 8 000 units
    https://www.bioz.com/result/bst large fragment polymerase/product/New England Biolabs
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    bst large fragment polymerase - by Bioz Stars, 2020-09
    97/100 stars

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    Related Articles

    Clone Assay:

    Article Title: Novel Bioluminescent Quantitative Detection of Nucleic Acid Amplification in Real-Time
    Article Snippet: .. Materials and reagents Unless otherwise noted, chemicals were purchased from Sigma with the exception of luciferin potassium salt (LH2 ; Europa Biotech, Ely, UK), UltraGlow firefly luciferase (UGrLuc; Promega, WI, USA), adenosine-5′-O-phosphosulphate (APS; Biolog Life Science Institute, Bremen, Germany), Bst DNA polymerase large fragment (Bst) and ThermoPol buffer (New England Biolabs, MA, USA), QuantiTech SYBR Green PCR kit (Qiagen, Hilden, Germany), cloned AMV reverse transcriptase and PicoGreen dsDNA Quantitation kit (Invitrogen, CA, USA). ..

    Luciferase:

    Article Title: Novel Bioluminescent Quantitative Detection of Nucleic Acid Amplification in Real-Time
    Article Snippet: .. Materials and reagents Unless otherwise noted, chemicals were purchased from Sigma with the exception of luciferin potassium salt (LH2 ; Europa Biotech, Ely, UK), UltraGlow firefly luciferase (UGrLuc; Promega, WI, USA), adenosine-5′-O-phosphosulphate (APS; Biolog Life Science Institute, Bremen, Germany), Bst DNA polymerase large fragment (Bst) and ThermoPol buffer (New England Biolabs, MA, USA), QuantiTech SYBR Green PCR kit (Qiagen, Hilden, Germany), cloned AMV reverse transcriptase and PicoGreen dsDNA Quantitation kit (Invitrogen, CA, USA). ..

    Incubation:

    Article Title: Iterative Epigenomic Analyses in the Same Single Cell
    Article Snippet: .. Four l of 10 x ThermoPol buffer (New England BioLabs) and dNTPs (total 1.4 mM) were added, and then the combined solution was heated at 94°C for 3 min and placed on ice for 2 min. Bst large fragment (2 μl/tube, M0275L, New England BioLabs) was added and incubated at 10°C for 45secs, 20°C for 45secs, 30°C for 45secs, 40°C for 45secs, 50°C for 45secs, 65°C for 2mins and 94°C for 20s. .. After quenching on ice, Bst large fragment (2 ul/tube) was added and then incubated at 10°C for 45secs, 20°C for 45secs, 30°C for 45secs, 40°C for 45secs, 50°C for 45secs, 65°C for 2mins and 94°C for 20s.

    SYBR Green Assay:

    Article Title: Novel Bioluminescent Quantitative Detection of Nucleic Acid Amplification in Real-Time
    Article Snippet: .. Materials and reagents Unless otherwise noted, chemicals were purchased from Sigma with the exception of luciferin potassium salt (LH2 ; Europa Biotech, Ely, UK), UltraGlow firefly luciferase (UGrLuc; Promega, WI, USA), adenosine-5′-O-phosphosulphate (APS; Biolog Life Science Institute, Bremen, Germany), Bst DNA polymerase large fragment (Bst) and ThermoPol buffer (New England Biolabs, MA, USA), QuantiTech SYBR Green PCR kit (Qiagen, Hilden, Germany), cloned AMV reverse transcriptase and PicoGreen dsDNA Quantitation kit (Invitrogen, CA, USA). ..

    Quantitation Assay:

    Article Title: Novel Bioluminescent Quantitative Detection of Nucleic Acid Amplification in Real-Time
    Article Snippet: .. Materials and reagents Unless otherwise noted, chemicals were purchased from Sigma with the exception of luciferin potassium salt (LH2 ; Europa Biotech, Ely, UK), UltraGlow firefly luciferase (UGrLuc; Promega, WI, USA), adenosine-5′-O-phosphosulphate (APS; Biolog Life Science Institute, Bremen, Germany), Bst DNA polymerase large fragment (Bst) and ThermoPol buffer (New England Biolabs, MA, USA), QuantiTech SYBR Green PCR kit (Qiagen, Hilden, Germany), cloned AMV reverse transcriptase and PicoGreen dsDNA Quantitation kit (Invitrogen, CA, USA). ..

    Amplification:

    Article Title: Real-Time Reverse Transcription Loop-Mediated Isothermal Amplification for Rapid Detection of West Nile Virus
    Article Snippet: .. The RT-LAMP reaction was carried out in a 25-μl total reaction mixture volume with a Loopamp DNA amplification kit (Eiken Chemical Co. Ltd.) containing 50 pmol each of inner primers FIP and BIP, 5 pmol each of outer primers F3 and B3, 25 pmol each of loop primers loop F and loop B, 1,400 μM each deoxynucleoside triphosphate, 0.6 M betaine (Sigma Chemical Co., St. Louis, Mo.), 40 mM Tris-HCl (pH 8.8), 20 mM KCl, 20 mM (NH4 )2 SO4 , 8 mM MgSO4 , 0.1% Triton X-100, 0.125 U of avian myeloblastosis virus (AMV) RTase (Invitrogen), 8 U of Bst DNA polymerase (large fragment; New England Biolabs), and the specified amounts of target RNA. .. The mixture was incubated at 63°C for 60 min in a heating block and then heated at 80°C for 2 min to terminate the reaction.

    Activity Assay:

    Article Title: COVID-19 Infection Diagnosis: Potential Impact of Isothermal Amplification Technology to Reduce Community Transmission of SARS-CoV-2
    Article Snippet: .. Essentially, LAMP uses a Bst DNA polymerase with strand-displacement activity, coupled with two inner primers (FIP, BIP) and outer primers (F3, B3) that recognizes six separate regions on a DNA template. .. Additional loop primers (LF, LB) may be added to speed up the reaction by binding to and amplifying newly formed loop amplicons in the reaction.

    Lamp Assay:

    Article Title: Loop-mediated isothermal amplification (LAMP) assays for the species-specific detection of Eimeria that infect chickens
    Article Snippet: .. LAMP assay Each LAMP reaction was performed in a final volume of 25 μl containing 8 U Bst DNA polymerase (large fragment; New England Biolabs) in 1 × ThermoPol Reaction buffer (20 mM Tris-HCl, 10 mM KCl, 10 mM (NH4 )2 SO4 , 2 mM MgSO4 and 0.1% Triton X-100; New England Biolabs) supplemented with 2 mM MgCl2 , 1 M betaine and 400 μM of each dNTP. ..

    Polymerase Chain Reaction:

    Article Title: Novel Bioluminescent Quantitative Detection of Nucleic Acid Amplification in Real-Time
    Article Snippet: .. Materials and reagents Unless otherwise noted, chemicals were purchased from Sigma with the exception of luciferin potassium salt (LH2 ; Europa Biotech, Ely, UK), UltraGlow firefly luciferase (UGrLuc; Promega, WI, USA), adenosine-5′-O-phosphosulphate (APS; Biolog Life Science Institute, Bremen, Germany), Bst DNA polymerase large fragment (Bst) and ThermoPol buffer (New England Biolabs, MA, USA), QuantiTech SYBR Green PCR kit (Qiagen, Hilden, Germany), cloned AMV reverse transcriptase and PicoGreen dsDNA Quantitation kit (Invitrogen, CA, USA). ..

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    New England Biolabs bst dna polymerase large fragment
    Detection of PSA mRNA by reverse transcription-coupled LAMP (RT-LAMP). Various numbers of LNCaP cells were mixed with 10 6 PSA-non-producing K562 cells and total RNA was extracted. RT-LAMP was carried out in the same reaction mixture as for M13mp18 <t>DNA</t> amplification except that 1.6 µM each PSAFIP and PSABIP, 0.2 µM each PSAF3 and PSAB3, 0.8 M betaine, 5 mM DTT, 16 U <t>Bst</t> polymerase, 100 U ReverTra Ace (Toyobo) and 5 µg of extracted RNA were used. All the above components were mixed at once on ice and were incubated at 65°C for 45 min. The products were electrophoresed in 2% agarose gel followed by SYBR Green I staining. + and –, RT-LAMP carried out in the presence and absence of Bst DNA polymerase or ReverTra Ace, respectively. Lanes 8 and 9, the same products (1/5 vol) as in lanes 6 and 7, respectively, but digested with Sau 3AI; lane M, 100 bp ladder (New England Biolabs).
    Bst Dna Polymerase Large Fragment, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 98/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bst dna polymerase large fragment/product/New England Biolabs
    Average 98 stars, based on 18 article reviews
    Price from $9.99 to $1999.99
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    91
    New England Biolabs general information bst dna polymerase large fragment
    Verification of UIMA using different <t>DNA</t> 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 <t>Bst</t> 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 .
    General Information Bst Dna Polymerase Large Fragment, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/general information bst dna polymerase large fragment/product/New England Biolabs
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    99
    New England Biolabs strand displacing bst dna polymerase enzyme
    mtDNA hybridisation enrichment protocol. mtDNA baits were prepared using 2 × 8 kb mtDNA long-range PCR products (spanning 16,569 base pairs), generated using a <t>DNA</t> extract from a present-day sample (of a known haplotype) and the Roche LR Expand PCR kit. The products were fragmented by physical shearing to create 200 bp to 600 bp fragments prior to end labelling with biotin. The DNA library was prepared as follows. Damaged DNA leaves 5′ and 3′ overhangs. T4 DNA polymerase was used to polish the DNA by creating blunt ends and T4 PNK phosphorylated 5′ ends, which is required for adaptor ligation. T4 ligase attached universal hybridisation adaptors (Uni-hyb A and Uni-hyb B) to the phosphorylated ends. Klenow polymerase filled in the short-arm adaptor ligation to create double-stranded adaptors (through the use of deoxyribonucleotide triphosphates - dNTPs). Adaptor complementary primers and Taq polymerase amplified the entire library to immortalise the sample. Single-stranded probe DNA was mixed with single-stranded library DNA and left to hybridise overnight (in the presence of blocking oligos). Biotinylated probe and bound library DNA were fixed to streptavidin beads on a magnetic rack, and non-specific or weakly bound library DNA was washed away through a series of three stringency washes (by increasing temperature and decreasing salt concentration) from the library–probe–streptavidin interaction. The single-stranded library DNA was converted to double stranded DNA and eluted from the probe–streptavidin interaction using the <t>Bst</t> strand-displacing enzyme (in the presence of dNTPs). Bst recognises nicks in the template and displaces library DNA into solution. Probe DNA remained bound to the magnet. Eluted library DNA was enriched through low cycle PCR, using adaptor complementary primers. Library DNA was then prepared for next-generation sequencing. mtDNA, mitochondrial DNA; PCR, polymerase chain reaction.
    Strand Displacing Bst Dna Polymerase Enzyme, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/strand displacing bst dna polymerase enzyme/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    strand displacing bst dna polymerase enzyme - by Bioz Stars, 2020-09
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    Detection of PSA mRNA by reverse transcription-coupled LAMP (RT-LAMP). Various numbers of LNCaP cells were mixed with 10 6 PSA-non-producing K562 cells and total RNA was extracted. RT-LAMP was carried out in the same reaction mixture as for M13mp18 DNA amplification except that 1.6 µM each PSAFIP and PSABIP, 0.2 µM each PSAF3 and PSAB3, 0.8 M betaine, 5 mM DTT, 16 U Bst polymerase, 100 U ReverTra Ace (Toyobo) and 5 µg of extracted RNA were used. All the above components were mixed at once on ice and were incubated at 65°C for 45 min. The products were electrophoresed in 2% agarose gel followed by SYBR Green I staining. + and –, RT-LAMP carried out in the presence and absence of Bst DNA polymerase or ReverTra Ace, respectively. Lanes 8 and 9, the same products (1/5 vol) as in lanes 6 and 7, respectively, but digested with Sau 3AI; lane M, 100 bp ladder (New England Biolabs).

    Journal: Nucleic Acids Research

    Article Title: Loop-mediated isothermal amplification of DNA

    doi:

    Figure Lengend Snippet: Detection of PSA mRNA by reverse transcription-coupled LAMP (RT-LAMP). Various numbers of LNCaP cells were mixed with 10 6 PSA-non-producing K562 cells and total RNA was extracted. RT-LAMP was carried out in the same reaction mixture as for M13mp18 DNA amplification except that 1.6 µM each PSAFIP and PSABIP, 0.2 µM each PSAF3 and PSAB3, 0.8 M betaine, 5 mM DTT, 16 U Bst polymerase, 100 U ReverTra Ace (Toyobo) and 5 µg of extracted RNA were used. All the above components were mixed at once on ice and were incubated at 65°C for 45 min. The products were electrophoresed in 2% agarose gel followed by SYBR Green I staining. + and –, RT-LAMP carried out in the presence and absence of Bst DNA polymerase or ReverTra Ace, respectively. Lanes 8 and 9, the same products (1/5 vol) as in lanes 6 and 7, respectively, but digested with Sau 3AI; lane M, 100 bp ladder (New England Biolabs).

    Article Snippet: The mixture was heated at 95°C for 5 min, then chilled on ice, 8 U Bst DNA polymerase large fragment (New England Biolabs) were added, followed by incubation at 65°C for 1 h and heating at 80°C for 10 min to terminate the reaction.

    Techniques: Amplification, Incubation, Agarose Gel Electrophoresis, SYBR Green Assay, Staining

    Restriction analysis and Southern blot hybridization of the amplified M13mp18 DNA. ( A ) Electrophoretic analysis of the LAMP amplified M13mp18 product. Six hundred copies of M13mp18 DNA were amplified by LAMP with the specific primers designed on the sequences shown in Figure 2 and run on a 2% agarose gel followed by SYBR Green I staining. Lane M, 100 bp ladder used as size marker (New England Biolabs); lane 1, M13mpl8 DNA digested with Pvu II; lane 2, LAMP without Bst DNA polymerase; lane 3, LAMP without target M13 DNA; lane 4, complete LAMP; lanes 5–7, complete LAMP products after digestion with Bam HI, Pst I and Pvu II, respectively (one fifth of the digests were loaded). (B–D) Southern blot analysis of the LAMP products. The 2% agarose gel shown in (A) was used for Southern blot hybridization with M13-281 DNA ( B ), M13-333 DNA ( C ) and M13BIP ( D ) as probes. ( E ) Alkaline agarose gel electrophoresis of the LAMP products. Lane m, λ DNA Hin dIII digests; lane 4, the same sample as in (A).

    Journal: Nucleic Acids Research

    Article Title: Loop-mediated isothermal amplification of DNA

    doi:

    Figure Lengend Snippet: Restriction analysis and Southern blot hybridization of the amplified M13mp18 DNA. ( A ) Electrophoretic analysis of the LAMP amplified M13mp18 product. Six hundred copies of M13mp18 DNA were amplified by LAMP with the specific primers designed on the sequences shown in Figure 2 and run on a 2% agarose gel followed by SYBR Green I staining. Lane M, 100 bp ladder used as size marker (New England Biolabs); lane 1, M13mpl8 DNA digested with Pvu II; lane 2, LAMP without Bst DNA polymerase; lane 3, LAMP without target M13 DNA; lane 4, complete LAMP; lanes 5–7, complete LAMP products after digestion with Bam HI, Pst I and Pvu II, respectively (one fifth of the digests were loaded). (B–D) Southern blot analysis of the LAMP products. The 2% agarose gel shown in (A) was used for Southern blot hybridization with M13-281 DNA ( B ), M13-333 DNA ( C ) and M13BIP ( D ) as probes. ( E ) Alkaline agarose gel electrophoresis of the LAMP products. Lane m, λ DNA Hin dIII digests; lane 4, the same sample as in (A).

    Article Snippet: The mixture was heated at 95°C for 5 min, then chilled on ice, 8 U Bst DNA polymerase large fragment (New England Biolabs) were added, followed by incubation at 65°C for 1 h and heating at 80°C for 10 min to terminate the reaction.

    Techniques: Southern Blot, Hybridization, Amplification, Agarose Gel Electrophoresis, SYBR Green Assay, Staining, Marker

    Amplification simulated diagram of the CPA and IMSA assays. A, Conditions and interval selection of assay optimization. B-C. Optimization of the incubation temperature, dNTPs concentration, Bst DNA polymerase concentration and incubation time for the CPA and IMSA assays, respectively. M: Standard DNA molecule marker; N: negative control. (The optimization items, corresponding conditions and units are marked in the figure, the best conditions highlighted with blue squares.).

    Journal: PLoS ONE

    Article Title: Establishment and application of isothermal amplification techniques for the detection of heat-stable I enterotoxin of enterotoxigenic Escherichia coli

    doi: 10.1371/journal.pone.0230881

    Figure Lengend Snippet: Amplification simulated diagram of the CPA and IMSA assays. A, Conditions and interval selection of assay optimization. B-C. Optimization of the incubation temperature, dNTPs concentration, Bst DNA polymerase concentration and incubation time for the CPA and IMSA assays, respectively. M: Standard DNA molecule marker; N: negative control. (The optimization items, corresponding conditions and units are marked in the figure, the best conditions highlighted with blue squares.).

    Article Snippet: The CPA assay was conducted in a reaction mixture of 30 μL total volume containing 6 μL 1s (10 mM), 1.5 μL 2a/3a (10 mM), 0.5 μL 4s/5a (10 mM), 2.5 μL (1.0 mM) dNTPs (Takara Bio, Dalian, CN), 3 μL 10 × Thermol-Pol reaction buffer, 1.5 μL Bst DNA polymerase large fragment (New England Biolabs, Ipswich, MA, USA), 3 μL (1 mM) MgCl2 (Sigma-Aldrich, St. Louis, MO, USA), 2 μL (0.8 M) betaine (Sigma-Aldrich) and 2 μL of an appropriate concentration of DNA template (E . coli C83920).

    Techniques: Amplification, Selection, Incubation, Concentration Assay, Marker, Negative Control

    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 .

    Journal: Scientific Reports

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

    doi: 10.1038/s41598-017-13324-0

    Figure Lengend 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 .

    Article Snippet: General information Bst DNA polymerase Large fragment (Bst LF), Bst 2.0 DNA polymerase (Bst 2.0), Bst 2.0 WarmStart DNA polymerase (Bst 2.0 WS), Bst 3.0 DNA polymerase (Bst 3.0), Klenow fragment polymerase (Klenow), Klenow fragment exo- polymerase (Klenow (exo-)), Vent exo- DNA polymerase (Vent (exo-)), and dNTP Mix were purchased from New England Biolabs.

    Techniques: 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 .

    Journal: Scientific Reports

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

    doi: 10.1038/s41598-017-13324-0

    Figure Lengend 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 .

    Article Snippet: General information Bst DNA polymerase Large fragment (Bst LF), Bst 2.0 DNA polymerase (Bst 2.0), Bst 2.0 WarmStart DNA polymerase (Bst 2.0 WS), Bst 3.0 DNA polymerase (Bst 3.0), Klenow fragment polymerase (Klenow), Klenow fragment exo- polymerase (Klenow (exo-)), Vent exo- DNA polymerase (Vent (exo-)), and dNTP Mix were purchased from New England Biolabs.

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

    mtDNA hybridisation enrichment protocol. mtDNA baits were prepared using 2 × 8 kb mtDNA long-range PCR products (spanning 16,569 base pairs), generated using a DNA extract from a present-day sample (of a known haplotype) and the Roche LR Expand PCR kit. The products were fragmented by physical shearing to create 200 bp to 600 bp fragments prior to end labelling with biotin. The DNA library was prepared as follows. Damaged DNA leaves 5′ and 3′ overhangs. T4 DNA polymerase was used to polish the DNA by creating blunt ends and T4 PNK phosphorylated 5′ ends, which is required for adaptor ligation. T4 ligase attached universal hybridisation adaptors (Uni-hyb A and Uni-hyb B) to the phosphorylated ends. Klenow polymerase filled in the short-arm adaptor ligation to create double-stranded adaptors (through the use of deoxyribonucleotide triphosphates - dNTPs). Adaptor complementary primers and Taq polymerase amplified the entire library to immortalise the sample. Single-stranded probe DNA was mixed with single-stranded library DNA and left to hybridise overnight (in the presence of blocking oligos). Biotinylated probe and bound library DNA were fixed to streptavidin beads on a magnetic rack, and non-specific or weakly bound library DNA was washed away through a series of three stringency washes (by increasing temperature and decreasing salt concentration) from the library–probe–streptavidin interaction. The single-stranded library DNA was converted to double stranded DNA and eluted from the probe–streptavidin interaction using the Bst strand-displacing enzyme (in the presence of dNTPs). Bst recognises nicks in the template and displaces library DNA into solution. Probe DNA remained bound to the magnet. Eluted library DNA was enriched through low cycle PCR, using adaptor complementary primers. Library DNA was then prepared for next-generation sequencing. mtDNA, mitochondrial DNA; PCR, polymerase chain reaction.

    Journal: Investigative Genetics

    Article Title: DNA capture and next-generation sequencing can recover whole mitochondrial genomes from highly degraded samples for human identification

    doi: 10.1186/2041-2223-4-26

    Figure Lengend Snippet: mtDNA hybridisation enrichment protocol. mtDNA baits were prepared using 2 × 8 kb mtDNA long-range PCR products (spanning 16,569 base pairs), generated using a DNA extract from a present-day sample (of a known haplotype) and the Roche LR Expand PCR kit. The products were fragmented by physical shearing to create 200 bp to 600 bp fragments prior to end labelling with biotin. The DNA library was prepared as follows. Damaged DNA leaves 5′ and 3′ overhangs. T4 DNA polymerase was used to polish the DNA by creating blunt ends and T4 PNK phosphorylated 5′ ends, which is required for adaptor ligation. T4 ligase attached universal hybridisation adaptors (Uni-hyb A and Uni-hyb B) to the phosphorylated ends. Klenow polymerase filled in the short-arm adaptor ligation to create double-stranded adaptors (through the use of deoxyribonucleotide triphosphates - dNTPs). Adaptor complementary primers and Taq polymerase amplified the entire library to immortalise the sample. Single-stranded probe DNA was mixed with single-stranded library DNA and left to hybridise overnight (in the presence of blocking oligos). Biotinylated probe and bound library DNA were fixed to streptavidin beads on a magnetic rack, and non-specific or weakly bound library DNA was washed away through a series of three stringency washes (by increasing temperature and decreasing salt concentration) from the library–probe–streptavidin interaction. The single-stranded library DNA was converted to double stranded DNA and eluted from the probe–streptavidin interaction using the Bst strand-displacing enzyme (in the presence of dNTPs). Bst recognises nicks in the template and displaces library DNA into solution. Probe DNA remained bound to the magnet. Eluted library DNA was enriched through low cycle PCR, using adaptor complementary primers. Library DNA was then prepared for next-generation sequencing. mtDNA, mitochondrial DNA; PCR, polymerase chain reaction.

    Article Snippet: The strand-displacing Bst DNA polymerase enzyme (large fragment, New England Biolabs) was used to release library DNA from the DNA-capture probe (immobilised to beads on the magnet).

    Techniques: Hybridization, Polymerase Chain Reaction, Generated, Ligation, Amplification, Blocking Assay, Concentration Assay, Next-Generation Sequencing