bst  (New England Biolabs)


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    Structured Review

    New England Biolabs bst
    The SMART assay. ( a ) Specific probes hybridise with the target to form a three-way junction (3WJ), assisted by facilitator probes (f1 f2). The 3WJ initially contains a single-stranded, inactive T7 RNA polymerase promoter sequence. The promoter is made double stranded (active) by extension (by <t>Bst</t> <t>DNA</t> polymerase) off the 3' of the extension probe, leading to the generation of large amounts of RNA signal (by T7 RNA polymerase), which may itself be amplified if required. ( b ) Detection of RNA signal by ELOSA (Enzyme Linked OligoSorbant Assay). The assay uses 2 specific probes: a biotinylated capture probe and enzyme (Alkaline phosphatase, AP) linked detection probe. Non-specific nucleic acid and 3WJ probes are removed, following binding in a streptavidin coated well, and RNA signal is detected via a colour change. Quantification of signal takes place in a 96 well plate, allowing multiple samples to be analysed simultaneously.
    Bst, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Detection of virus mRNA within infected host cells using an isothermal nucleic acid amplification assay: marine cyanophage gene expression within Synechococcus sp"

    Article Title: Detection of virus mRNA within infected host cells using an isothermal nucleic acid amplification assay: marine cyanophage gene expression within Synechococcus sp

    Journal: Virology Journal

    doi: 10.1186/1743-422X-4-52

    The SMART assay. ( a ) Specific probes hybridise with the target to form a three-way junction (3WJ), assisted by facilitator probes (f1 f2). The 3WJ initially contains a single-stranded, inactive T7 RNA polymerase promoter sequence. The promoter is made double stranded (active) by extension (by Bst DNA polymerase) off the 3' of the extension probe, leading to the generation of large amounts of RNA signal (by T7 RNA polymerase), which may itself be amplified if required. ( b ) Detection of RNA signal by ELOSA (Enzyme Linked OligoSorbant Assay). The assay uses 2 specific probes: a biotinylated capture probe and enzyme (Alkaline phosphatase, AP) linked detection probe. Non-specific nucleic acid and 3WJ probes are removed, following binding in a streptavidin coated well, and RNA signal is detected via a colour change. Quantification of signal takes place in a 96 well plate, allowing multiple samples to be analysed simultaneously.
    Figure Legend Snippet: The SMART assay. ( a ) Specific probes hybridise with the target to form a three-way junction (3WJ), assisted by facilitator probes (f1 f2). The 3WJ initially contains a single-stranded, inactive T7 RNA polymerase promoter sequence. The promoter is made double stranded (active) by extension (by Bst DNA polymerase) off the 3' of the extension probe, leading to the generation of large amounts of RNA signal (by T7 RNA polymerase), which may itself be amplified if required. ( b ) Detection of RNA signal by ELOSA (Enzyme Linked OligoSorbant Assay). The assay uses 2 specific probes: a biotinylated capture probe and enzyme (Alkaline phosphatase, AP) linked detection probe. Non-specific nucleic acid and 3WJ probes are removed, following binding in a streptavidin coated well, and RNA signal is detected via a colour change. Quantification of signal takes place in a 96 well plate, allowing multiple samples to be analysed simultaneously.

    Techniques Used: Sequencing, Amplification, Binding Assay

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    New England Biolabs bst dna polymerase large fragment
    qPCR performed on output from <t>BST</t> LF <t>DNA</t> amplification (15 min) targeting 100 copies of P. knowlesi using only the F3 and B3 primers from the P.KNO-LAU primer set. Activity assay, A (i) commercial BST LF (8 U); (ii) Si-R5 2 -mCh-FL-BST LF in ThermoPol® buffer with added Mg 2+ ; (iii) 0.2 × isothermal amplification buffer with added Mn 2+ and Mg 2+ ; (iv) Si-R5 2 -mCh-FL-BST LF immobilised in PBS and used in ThermoPol® buffer with added Mg 2+ . B Comparing the recombinant BST LF on silica in ThermoPol® buffer, supplemented with different concentrations of Mn 2+ and Mg 2+ . 1U of BST LF is defined as the amount of enzyme requi red to incorporate 10 nmol of dNTP in 30 min at 65 °C.
    Bst Dna Polymerase Large Fragment, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    qPCR performed on output from BST LF DNA amplification (15 min) targeting 100 copies of P. knowlesi using only the F3 and B3 primers from the P.KNO-LAU primer set. Activity assay, A (i) commercial BST LF (8 U); (ii) Si-R5 2 -mCh-FL-BST LF in ThermoPol® buffer with added Mg 2+ ; (iii) 0.2 × isothermal amplification buffer with added Mn 2+ and Mg 2+ ; (iv) Si-R5 2 -mCh-FL-BST LF immobilised in PBS and used in ThermoPol® buffer with added Mg 2+ . B Comparing the recombinant BST LF on silica in ThermoPol® buffer, supplemented with different concentrations of Mn 2+ and Mg 2+ . 1U of BST LF is defined as the amount of enzyme requi red to incorporate 10 nmol of dNTP in 30 min at 65 °C.

    Journal: Analytical and Bioanalytical Chemistry

    Article Title: Analysis and validation of silica-immobilised BST polymerase in loop-mediated isothermal amplification (LAMP) for malaria diagnosis

    doi: 10.1007/s00216-022-04131-2

    Figure Lengend Snippet: qPCR performed on output from BST LF DNA amplification (15 min) targeting 100 copies of P. knowlesi using only the F3 and B3 primers from the P.KNO-LAU primer set. Activity assay, A (i) commercial BST LF (8 U); (ii) Si-R5 2 -mCh-FL-BST LF in ThermoPol® buffer with added Mg 2+ ; (iii) 0.2 × isothermal amplification buffer with added Mn 2+ and Mg 2+ ; (iv) Si-R5 2 -mCh-FL-BST LF immobilised in PBS and used in ThermoPol® buffer with added Mg 2+ . B Comparing the recombinant BST LF on silica in ThermoPol® buffer, supplemented with different concentrations of Mn 2+ and Mg 2+ . 1U of BST LF is defined as the amount of enzyme requi red to incorporate 10 nmol of dNTP in 30 min at 65 °C.

    Article Snippet: Q5 high-fidelity DNA polymerase, Klenow fragment, BST DNA Polymerase Large Fragment, NEBuffer™ 2, isothermal amplification buffer II, ThermoPol® reaction buffer, magnesium sulfate (MgSO4 ), deoxynucleotide solution mix, BbsI-HF®, MfeI-HF®, rCutSmart™ buffer, 6 × DNA loading dye, nuclease-free water, NEB® Turbo Competent E. coli (High Efficiency) and BL21 (DE3) competent E. coli were obtained from New England Biolabs.

    Techniques: Real-time Polymerase Chain Reaction, Amplification, Activity Assay, Recombinant

    A – D 2% agarose gel electrophoresis of 90-min limit of detection LAMP using 8U of commercial BST LF for the malaria primer sets showing false positives A P.FAL-LAU, B P.OVA-LAU, C P.VIV-HAN, D P.OVA-HAN. The number above each gel refers to the number of copies of plasmid DNA. E – H Restriction analysis of the true positive (TP) and false positive (FP). The products were run on a 2.5% agarose gel. E BbsI-HF® digestion of P.FAL-LAU products (expected band size of 224 bp). F BbsI-HF® digestion of P.OVA-LAU products (expected band size of 276 bp). G MfeI-HF® digestion of P.VIV-HAN products (expected band size of 219 bp). H MfeI-HF® digestion of P.OVA-HAN products (expected band size of 240 bp)

    Journal: Analytical and Bioanalytical Chemistry

    Article Title: Analysis and validation of silica-immobilised BST polymerase in loop-mediated isothermal amplification (LAMP) for malaria diagnosis

    doi: 10.1007/s00216-022-04131-2

    Figure Lengend Snippet: A – D 2% agarose gel electrophoresis of 90-min limit of detection LAMP using 8U of commercial BST LF for the malaria primer sets showing false positives A P.FAL-LAU, B P.OVA-LAU, C P.VIV-HAN, D P.OVA-HAN. The number above each gel refers to the number of copies of plasmid DNA. E – H Restriction analysis of the true positive (TP) and false positive (FP). The products were run on a 2.5% agarose gel. E BbsI-HF® digestion of P.FAL-LAU products (expected band size of 224 bp). F BbsI-HF® digestion of P.OVA-LAU products (expected band size of 276 bp). G MfeI-HF® digestion of P.VIV-HAN products (expected band size of 219 bp). H MfeI-HF® digestion of P.OVA-HAN products (expected band size of 240 bp)

    Article Snippet: Q5 high-fidelity DNA polymerase, Klenow fragment, BST DNA Polymerase Large Fragment, NEBuffer™ 2, isothermal amplification buffer II, ThermoPol® reaction buffer, magnesium sulfate (MgSO4 ), deoxynucleotide solution mix, BbsI-HF®, MfeI-HF®, rCutSmart™ buffer, 6 × DNA loading dye, nuclease-free water, NEB® Turbo Competent E. coli (High Efficiency) and BL21 (DE3) competent E. coli were obtained from New England Biolabs.

    Techniques: Agarose Gel Electrophoresis, Plasmid Preparation

    A Percentage of total protein binding from cell lysate to 5 mg of silica based on NanoDrop estimation of protein concentration in the lysate. B Comparison of R5 2 -mCh-H10-BST LF loading efficiency (%) with the amount of BST LF /mg of silica ( n = 3). C Agarose gel electrophoresis of LAMP products from 10 7 copies of P. malariae 18S rRNA plasmid DNA using the P.MAL-LAU primer set (Table 1 ) and using 0.4 mg of silica with immobilised BST from cell lysate: lanes 1 and 2, 1250 µg of cell lysate + 15 mg of silica giving 2.5 µg/mg R5 2 -mCh-H10-BST LF /silica; lanes 3 and 4, 1250 µg of cell lysate + 5 mg of silica giving 4.15 µg/mg R5 2 -mCh-H10-BST LF /silica. LAMP was performed for 90 min. D Agarose gel electrophoresis of the DNA from the cell lysate (5 µL) containing R5 2 -mCh-H10-BST LF showing the presence of native DNA fragments from 100–10,000 base pairs

    Journal: Analytical and Bioanalytical Chemistry

    Article Title: Analysis and validation of silica-immobilised BST polymerase in loop-mediated isothermal amplification (LAMP) for malaria diagnosis

    doi: 10.1007/s00216-022-04131-2

    Figure Lengend Snippet: A Percentage of total protein binding from cell lysate to 5 mg of silica based on NanoDrop estimation of protein concentration in the lysate. B Comparison of R5 2 -mCh-H10-BST LF loading efficiency (%) with the amount of BST LF /mg of silica ( n = 3). C Agarose gel electrophoresis of LAMP products from 10 7 copies of P. malariae 18S rRNA plasmid DNA using the P.MAL-LAU primer set (Table 1 ) and using 0.4 mg of silica with immobilised BST from cell lysate: lanes 1 and 2, 1250 µg of cell lysate + 15 mg of silica giving 2.5 µg/mg R5 2 -mCh-H10-BST LF /silica; lanes 3 and 4, 1250 µg of cell lysate + 5 mg of silica giving 4.15 µg/mg R5 2 -mCh-H10-BST LF /silica. LAMP was performed for 90 min. D Agarose gel electrophoresis of the DNA from the cell lysate (5 µL) containing R5 2 -mCh-H10-BST LF showing the presence of native DNA fragments from 100–10,000 base pairs

    Article Snippet: Q5 high-fidelity DNA polymerase, Klenow fragment, BST DNA Polymerase Large Fragment, NEBuffer™ 2, isothermal amplification buffer II, ThermoPol® reaction buffer, magnesium sulfate (MgSO4 ), deoxynucleotide solution mix, BbsI-HF®, MfeI-HF®, rCutSmart™ buffer, 6 × DNA loading dye, nuclease-free water, NEB® Turbo Competent E. coli (High Efficiency) and BL21 (DE3) competent E. coli were obtained from New England Biolabs.

    Techniques: Protein Binding, Protein Concentration, Agarose Gel Electrophoresis, Plasmid Preparation

    Detection of Skp2 amplification in NSCLC samples following whole genome amplification by Bst DNA polymerase . The ratios of Skp2 to PIK3R1 gene were maintained in Bst amplified vs. non-amplified NSCLC samples. Error bars represent SD.

    Journal: BMC Genomics

    Article Title: Large fragment Bst DNA polymerase for whole genome amplification of DNA from formalin-fixed paraffin-embedded tissues

    doi: 10.1186/1471-2164-7-312

    Figure Lengend Snippet: Detection of Skp2 amplification in NSCLC samples following whole genome amplification by Bst DNA polymerase . The ratios of Skp2 to PIK3R1 gene were maintained in Bst amplified vs. non-amplified NSCLC samples. Error bars represent SD.

    Article Snippet: The reaction mixture was then brought up to 50 μL with 400 μM dNTPs in 1× ThermoPol buffer, 0.35 units/μL Bst DNA polymerase, large fragment (New England Biolabs, Ipswich, MA) and 4% final concentration of DMSO.

    Techniques: Amplification, Whole Genome Amplification

    Gel electrophoresis of Bst DNA polymerase amplification products . From left to right: (1) Lambda DNA-Hind III digested ladder; FFPE samples: (2, 3) normal lung 3 4; (4, 5) neuroblastoma xenografts LAN-5 SK-N-BE (2) and (6, 7) NSCLC 3 4; (8) Commercial DNA; (9) Negative control. Samples were analyzed in 0.5% agarose gel, stained with SYBR-green II. 10% by volume of the amplification product was used for the gel electrophoresis.

    Journal: BMC Genomics

    Article Title: Large fragment Bst DNA polymerase for whole genome amplification of DNA from formalin-fixed paraffin-embedded tissues

    doi: 10.1186/1471-2164-7-312

    Figure Lengend Snippet: Gel electrophoresis of Bst DNA polymerase amplification products . From left to right: (1) Lambda DNA-Hind III digested ladder; FFPE samples: (2, 3) normal lung 3 4; (4, 5) neuroblastoma xenografts LAN-5 SK-N-BE (2) and (6, 7) NSCLC 3 4; (8) Commercial DNA; (9) Negative control. Samples were analyzed in 0.5% agarose gel, stained with SYBR-green II. 10% by volume of the amplification product was used for the gel electrophoresis.

    Article Snippet: The reaction mixture was then brought up to 50 μL with 400 μM dNTPs in 1× ThermoPol buffer, 0.35 units/μL Bst DNA polymerase, large fragment (New England Biolabs, Ipswich, MA) and 4% final concentration of DMSO.

    Techniques: Nucleic Acid Electrophoresis, Amplification, Lambda DNA Preparation, Formalin-fixed Paraffin-Embedded, Negative Control, Agarose Gel Electrophoresis, Staining, SYBR Green Assay

    N- myc gene content in Bst amplified vs. non-amplified neuroblastoma xenografts . For neuroblastoma xenografts, where N- myc gene is highly amplified, relative gene content in Bst amplified samples was comparable to the respective values in non-amplified samples and the representational distortion was negligible. Note: NBL-S is a neuroblastoma cell line that lacks N- myc amplification and appropriately the calculated copy numbers were 1.12 ± 0.03 for non-amplified DNA and 1.14 ± 0.35 for Bst amplified DNA. Error bars represent SD.

    Journal: BMC Genomics

    Article Title: Large fragment Bst DNA polymerase for whole genome amplification of DNA from formalin-fixed paraffin-embedded tissues

    doi: 10.1186/1471-2164-7-312

    Figure Lengend Snippet: N- myc gene content in Bst amplified vs. non-amplified neuroblastoma xenografts . For neuroblastoma xenografts, where N- myc gene is highly amplified, relative gene content in Bst amplified samples was comparable to the respective values in non-amplified samples and the representational distortion was negligible. Note: NBL-S is a neuroblastoma cell line that lacks N- myc amplification and appropriately the calculated copy numbers were 1.12 ± 0.03 for non-amplified DNA and 1.14 ± 0.35 for Bst amplified DNA. Error bars represent SD.

    Article Snippet: The reaction mixture was then brought up to 50 μL with 400 μM dNTPs in 1× ThermoPol buffer, 0.35 units/μL Bst DNA polymerase, large fragment (New England Biolabs, Ipswich, MA) and 4% final concentration of DMSO.

    Techniques: Amplification

    Mean amplification of DNA by Bst polymerase . All reactions started with 10 ng of target DNA. FFPE samples: Lung 1–5, neuroblastoma xenografts (LAN-5, NUB-7, SK-N-BE(2), NBL-S) and NSCLC 1–7. Intact DNA samples: FL (Frozen Lung) 1–4 and Positive C. (Control). Negative C. (Control) contained water in lieu of target DNA. For each sample the mean and SD of 2–6 independent experiments is shown.

    Journal: BMC Genomics

    Article Title: Large fragment Bst DNA polymerase for whole genome amplification of DNA from formalin-fixed paraffin-embedded tissues

    doi: 10.1186/1471-2164-7-312

    Figure Lengend Snippet: Mean amplification of DNA by Bst polymerase . All reactions started with 10 ng of target DNA. FFPE samples: Lung 1–5, neuroblastoma xenografts (LAN-5, NUB-7, SK-N-BE(2), NBL-S) and NSCLC 1–7. Intact DNA samples: FL (Frozen Lung) 1–4 and Positive C. (Control). Negative C. (Control) contained water in lieu of target DNA. For each sample the mean and SD of 2–6 independent experiments is shown.

    Article Snippet: The reaction mixture was then brought up to 50 μL with 400 μM dNTPs in 1× ThermoPol buffer, 0.35 units/μL Bst DNA polymerase, large fragment (New England Biolabs, Ipswich, MA) and 4% final concentration of DMSO.

    Techniques: Amplification, Formalin-fixed Paraffin-Embedded

    Overview of the experimental steps required to create and analyse a chromatin accessibility library. ( A ) Step 1: fungal mycelia pre-grown under specific conditions or isolated DNA ( in vitro controls) are processed as described in Materials and methods section and digested with MNase or restriction enzymes of choice. Step 2: digested DNA is blunt-ended and phosphorylated by subsequent treatment of the chromatin with Klenow fragment polymerase, T4 polynucleotide kinase. This step produces blunt-ended DNA fragments for ligation with adaptors. Step 3: DNA fragments are ligated with double-stranded adaptors A and B, originating from oligonucleotides Adaptor-A short and Adaptor-A long or Adaptor-B short and Adaptor-B long , where adaptor oligonucleotide B long is biotinylated for later retention on the streptavidin beads. In this step, fragments containing all adaptor combinations (A-A, A-B and B-B) are generated. Step 4: the ligation step leaves nicks at the 3′-terminus that are repaired by Bst polymerase treatment. Step 5: all fragments containing biotinylated adaptor B are captured on streptavidin-coated magnetic beads. At this step, adaptor A-A fragments are lost. Step 6: after a washing step, the retained fragments (adaptors A-B and B-B fragments) are denatured at 95°C. The denaturation step results in the release of single strands which exclusively carry A-B adaptor fragments. Step 7: the single-stranded A-B adaptor fragment library is amplified by a nested PCR approach to give the final A-B fragment library. The input and output fragment libraries are quality controlled by amplification with single A and B, as well as mixed A-B primers. Only the A-B primer mix should result in the amplification of fragments in the range of 200–1000 bp (see Panel B). Step 8: the resulting A-B adaptor fragment library is diluted and aliquots are used for analytical PCR amplifications for fragment size analysis of specific loci of interest. In the final analytical PCR step, either gene-specific or adaptor-specific primers can be labelled for subsequent capillary sequencer analysis. The chromatograms are finally analysed by image analysis software. ( B ) Example of quality control of A-B adaptor fragment libraries. Two input chromatin fragment libraries without adaptor ligation (lanes 1 and 2) are compared to two output libraries with adaptor ligation as described in Materials and methods section (lanes 3 and 4). Libraries originating from nitrate-grown cells (lanes 1 and 3) as well as from ammonium-grown cells (lanes 2 and 4) are shown as an example. M, DNA size marker.

    Journal: Nucleic Acids Research

    Article Title: A library-based method to rapidly analyse chromatin accessibility at multiple genomic regions

    doi: 10.1093/nar/gkp037

    Figure Lengend Snippet: Overview of the experimental steps required to create and analyse a chromatin accessibility library. ( A ) Step 1: fungal mycelia pre-grown under specific conditions or isolated DNA ( in vitro controls) are processed as described in Materials and methods section and digested with MNase or restriction enzymes of choice. Step 2: digested DNA is blunt-ended and phosphorylated by subsequent treatment of the chromatin with Klenow fragment polymerase, T4 polynucleotide kinase. This step produces blunt-ended DNA fragments for ligation with adaptors. Step 3: DNA fragments are ligated with double-stranded adaptors A and B, originating from oligonucleotides Adaptor-A short and Adaptor-A long or Adaptor-B short and Adaptor-B long , where adaptor oligonucleotide B long is biotinylated for later retention on the streptavidin beads. In this step, fragments containing all adaptor combinations (A-A, A-B and B-B) are generated. Step 4: the ligation step leaves nicks at the 3′-terminus that are repaired by Bst polymerase treatment. Step 5: all fragments containing biotinylated adaptor B are captured on streptavidin-coated magnetic beads. At this step, adaptor A-A fragments are lost. Step 6: after a washing step, the retained fragments (adaptors A-B and B-B fragments) are denatured at 95°C. The denaturation step results in the release of single strands which exclusively carry A-B adaptor fragments. Step 7: the single-stranded A-B adaptor fragment library is amplified by a nested PCR approach to give the final A-B fragment library. The input and output fragment libraries are quality controlled by amplification with single A and B, as well as mixed A-B primers. Only the A-B primer mix should result in the amplification of fragments in the range of 200–1000 bp (see Panel B). Step 8: the resulting A-B adaptor fragment library is diluted and aliquots are used for analytical PCR amplifications for fragment size analysis of specific loci of interest. In the final analytical PCR step, either gene-specific or adaptor-specific primers can be labelled for subsequent capillary sequencer analysis. The chromatograms are finally analysed by image analysis software. ( B ) Example of quality control of A-B adaptor fragment libraries. Two input chromatin fragment libraries without adaptor ligation (lanes 1 and 2) are compared to two output libraries with adaptor ligation as described in Materials and methods section (lanes 3 and 4). Libraries originating from nitrate-grown cells (lanes 1 and 3) as well as from ammonium-grown cells (lanes 2 and 4) are shown as an example. M, DNA size marker.

    Article Snippet: Fifteen microlitres of the adaptor ligated DNA were incubated for 30 min at 65°C in 1× ThermoPol Reaction Buffer (New England Biolabs), 8 µg Bovine Serum Albumin (BSA) (New England Biolabs), 20 nmol dNTPs and 16 U Bst DNA polymerase, Large Fragment (New England Biolabs) in a total volume of 20 µl.

    Techniques: Isolation, In Vitro, Ligation, Generated, Magnetic Beads, Amplification, Nested PCR, Polymerase Chain Reaction, Software, Marker

    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