tth endonuclease iv (New England Biolabs)

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Tth Endonuclease IV

Description:

Tth Endonuclease IV 500 units

Catalog Number:

m0294s

Price:

Size:

500 units

Category:

Deoxyribonucleases DNase

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New England Biolabs tth endonuclease iv

Tth Endonuclease IV 500 units
https://www.bioz.com/result/tth endonuclease iv/product/New England Biolabs
Average 79 stars, based on 1 article reviews
Price from $9.99 to $1999.99

tth endonuclease iv - by Bioz Stars, 2020-01

79/100 stars

Related Products / Commonly Used Together

bst 2.0 warmstart dna polymerase
deoxynucleotide triphosphate set
mgso4
iac oligonucleotides
isothermal amplification buffer

Images

1) Product Images from "Evaluation of an Internally Controlled Multiplex Tth Endonuclease Cleavage Loop-Mediated Isothermal Amplification (TEC-LAMP) Assay for the Detection of Bacterial Meningitis Pathogens"

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19020524

TEC-LAMP mechanism. ( A ) TEC-LAMP oligonucleotide components, Tth endonuclease IV enzyme and dsDNA template with oligonucleotide targets highlighted. ( B ) Temperature enabled dsDNA dissociation followed by primer and TEC primer/probe hybridization to corresponding targets. ( C ) Inner primer strand displacement extension, via Bst polymerase, forms dsDNA. Outer primer strand displacement extension dissociates this newly formed dsDNA, forming inner primer linked ssDNA. ( D ) The complementary sections of this newly formed inner primer linked ssDNA hybridize, forming loop structures. The abasic site of the TEC primer/probe is now in dsDNA form, and thus, cleaved by the Tth endonuclease IV enzyme. TEC primer/probe, inner and outer primers hybridize upstream of the stem loop structures. ( E ): Inner primer strand displacement extension forms dsDNA and displaces the downstream stem loop structures, fully dissociating the TEC primer/probe fluorophore and quencher, producing fluorescence. Outer primer strand displacement extension displaces the newly formed dsDNA, producing inner primer linked ssDNA. ( F ) The complementary sections at each end of the newly formed inner primer linked ssDNA hybridize and form loop structures. These double looped DNA templates are targeted by the TEC primer/probe, inner and loop primers, leading to rapid self-primed exponential amplification with increased cleavage and fluorescence events.

Figure Legend Snippet: TEC-LAMP mechanism. ( A ) TEC-LAMP oligonucleotide components, Tth endonuclease IV enzyme and dsDNA template with oligonucleotide targets highlighted. ( B ) Temperature enabled dsDNA dissociation followed by primer and TEC primer/probe hybridization to corresponding targets. ( C ) Inner primer strand displacement extension, via Bst polymerase, forms dsDNA. Outer primer strand displacement extension dissociates this newly formed dsDNA, forming inner primer linked ssDNA. ( D ) The complementary sections of this newly formed inner primer linked ssDNA hybridize, forming loop structures. The abasic site of the TEC primer/probe is now in dsDNA form, and thus, cleaved by the Tth endonuclease IV enzyme. TEC primer/probe, inner and outer primers hybridize upstream of the stem loop structures. ( E ): Inner primer strand displacement extension forms dsDNA and displaces the downstream stem loop structures, fully dissociating the TEC primer/probe fluorophore and quencher, producing fluorescence. Outer primer strand displacement extension displaces the newly formed dsDNA, producing inner primer linked ssDNA. ( F ) The complementary sections at each end of the newly formed inner primer linked ssDNA hybridize and form loop structures. These double looped DNA templates are targeted by the TEC primer/probe, inner and loop primers, leading to rapid self-primed exponential amplification with increased cleavage and fluorescence events.

Techniques Used: Hybridization, Fluorescence, Amplification

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.

Amplification:

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: .. 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. .. Reactions were performed for 60 × 1 min cycles at 67 °C in a LightCycler® 480 instrument II (Roche Diagnostics).

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
Article Snippet: 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. .. A color compensation file, generated as per LightCycler® 480 operator manual, was applied for correction of any channel-to-channel fluorescence cross-talk.

Fluorescence:

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: 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. .. The fluorescence detection channels used were 450–500 nm (Cyan), 495–520 nm (FAM), 535–565 nm (HEX) and 646–662 nm (Cy5), with fluorescent measurements recorded every cycle.