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    New England Biolabs triton x 100
    Ability of mutant Rep proteins to introduce ITR plasmid into AAVS1. Two micrograms of pCMVR78, mutant Rep expression plasmids, or blank vector was transfected into 2 × 10 5  293 cells/well in six-well plates along with 2 μg of pW1, harboring a  lacZ  expression cassette flanked by ITRs, by a standard calcium phosphate precipitation method. Twenty-four hours later, total cellular DNA was isolated and suspended finally in 200 μl of TE. PCR to detect site-specific integration was carried out as reported previously with minor modifications: 1 μl of isolated genomic DNA was subjected to a thermal cycling reaction in a 20-μl reaction mixture containing 1× thermophilic DNA polymerase buffer [10 mM KCl, 20 mM Tris-HCl (pH 8.8), 10 mM (NH 4 ) 2 SO 4 , 4 mM MgSO 4 , 0.1% Triton X-100 (NEB)], 1 μM 5′-CGGCCTCAGTGAGCGAGCGAGC and 5′-CGGGGAGGATCCGCTCAGAGGACA, and 2 U of Deep Vent Exo(−) DNA polymerase (NEB). The cycling conditions were 99°C for 1 min followed by 35 cycles of 99°C for 10 s and 72°C for 4 min. Ten microliters of the PCR mixture was transferred to a hybridization membrane (Hybond-N + ; Amersham) by using a dot blot apparatus and hybridized with a  32 P-labeled AAVS1 probe. The membranes were then analyzed on a BAS-1500 imaging analyzer. The assay was repeated at least four times. p, positive control for hybridization.
    Triton X 100, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 49 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Charged-to-Alanine Scanning Mutagenesis of the N-Terminal Half of Adeno-Associated Virus Type 2 Rep78 Protein"

    Article Title: Charged-to-Alanine Scanning Mutagenesis of the N-Terminal Half of Adeno-Associated Virus Type 2 Rep78 Protein

    Journal: Journal of Virology

    doi:

    Ability of mutant Rep proteins to introduce ITR plasmid into AAVS1. Two micrograms of pCMVR78, mutant Rep expression plasmids, or blank vector was transfected into 2 × 10 5  293 cells/well in six-well plates along with 2 μg of pW1, harboring a  lacZ  expression cassette flanked by ITRs, by a standard calcium phosphate precipitation method. Twenty-four hours later, total cellular DNA was isolated and suspended finally in 200 μl of TE. PCR to detect site-specific integration was carried out as reported previously with minor modifications: 1 μl of isolated genomic DNA was subjected to a thermal cycling reaction in a 20-μl reaction mixture containing 1× thermophilic DNA polymerase buffer [10 mM KCl, 20 mM Tris-HCl (pH 8.8), 10 mM (NH 4 ) 2 SO 4 , 4 mM MgSO 4 , 0.1% Triton X-100 (NEB)], 1 μM 5′-CGGCCTCAGTGAGCGAGCGAGC and 5′-CGGGGAGGATCCGCTCAGAGGACA, and 2 U of Deep Vent Exo(−) DNA polymerase (NEB). The cycling conditions were 99°C for 1 min followed by 35 cycles of 99°C for 10 s and 72°C for 4 min. Ten microliters of the PCR mixture was transferred to a hybridization membrane (Hybond-N + ; Amersham) by using a dot blot apparatus and hybridized with a  32 P-labeled AAVS1 probe. The membranes were then analyzed on a BAS-1500 imaging analyzer. The assay was repeated at least four times. p, positive control for hybridization.
    Figure Legend Snippet: Ability of mutant Rep proteins to introduce ITR plasmid into AAVS1. Two micrograms of pCMVR78, mutant Rep expression plasmids, or blank vector was transfected into 2 × 10 5 293 cells/well in six-well plates along with 2 μg of pW1, harboring a lacZ expression cassette flanked by ITRs, by a standard calcium phosphate precipitation method. Twenty-four hours later, total cellular DNA was isolated and suspended finally in 200 μl of TE. PCR to detect site-specific integration was carried out as reported previously with minor modifications: 1 μl of isolated genomic DNA was subjected to a thermal cycling reaction in a 20-μl reaction mixture containing 1× thermophilic DNA polymerase buffer [10 mM KCl, 20 mM Tris-HCl (pH 8.8), 10 mM (NH 4 ) 2 SO 4 , 4 mM MgSO 4 , 0.1% Triton X-100 (NEB)], 1 μM 5′-CGGCCTCAGTGAGCGAGCGAGC and 5′-CGGGGAGGATCCGCTCAGAGGACA, and 2 U of Deep Vent Exo(−) DNA polymerase (NEB). The cycling conditions were 99°C for 1 min followed by 35 cycles of 99°C for 10 s and 72°C for 4 min. Ten microliters of the PCR mixture was transferred to a hybridization membrane (Hybond-N + ; Amersham) by using a dot blot apparatus and hybridized with a 32 P-labeled AAVS1 probe. The membranes were then analyzed on a BAS-1500 imaging analyzer. The assay was repeated at least four times. p, positive control for hybridization.

    Techniques Used: Mutagenesis, Introduce, Plasmid Preparation, Expressing, Transfection, Isolation, Polymerase Chain Reaction, Hybridization, Dot Blot, Labeling, Imaging, Positive Control

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    Amplification:

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    Article Title: Evaluating the influence of life‐history characteristics on genetic structure: a comparison of small mammals inhabiting complex agricultural landscapes
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    Article Title: In vitro selection of an XNA aptamer capable of small-molecule recognition
    Article Snippet: Paragraph title: PCR amplification of ssDNA library ... 2 mM MgSO4 , 0.1% Triton X-100, pH 8.8, New England Biolabs).

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    Article Title: Discerning non-disjunction in Down syndrome patients by means of GluK1-(AGAT)n and D21S2055-(GATA)n microsatellites on chromosome 21
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    Article Title: Mutational Disruption of cis-Acting Replication Element 2C in Coxsackievirus B3 Leads to 5′-Terminal Genomic Deletions
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    Article Title: Development and application of a rapid and visual loop-mediated isothermal amplification for the detection of Sporisorium scitamineum in sugarcane
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    Positive Control:

    Article Title: Novel thermostable Y-family polymerases: applications for the PCR amplification of damaged or ancient DNAs
    Article Snippet: PCRs on undamaged DNA For the undamaged plasmid DNA template, 50 µl PCRs were set up containing standard ThermoPol reaction buffer [20 mM Tris–HCl, pH 8.8, 10 mM KCl, 10 mM (NH4 )2 SO4 , 2 mM MgSO4 , 0.1% Triton X-100] (New England Biolabs), 10 ng of circular pJM548 (Ste dpo4 -like in pGEM-T), 200 µM of each ultrapure dNTP and 1 µM of each primer; Ste_FNde5 and Ste_RBam1089 whose sequences are provided above. .. Aliquots containing 2.5 U of Taq DNA polymerase (New England Biolabs) were used as a positive control.

    Blocking Assay:

    Article Title: Strong-LAMP: A LAMP Assay for Strongyloides spp. Detection in Stool and Urine Samples. Towards the Diagnosis of Human Strongyloidiasis Starting from a Rodent Model
    Article Snippet: Thus, LAMP reactions mixtures (25 μL) contained 40 pmol of each FIP and BIP primers, 5 pmol of each F3 and B3 primers, 1.4 mM of each dNTP (Bioron), 1x ThermoPol Reaction Buffer -20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4 )2 SO4 , 2 mM MgSO4 , 0.1% Triton X-100; New England Biolabs, UK- (when using Bst Polymerase Large Fragment) or 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- (when using either Bst DNA polymerase 2.0 or Bst DNA polymerase 2.0 WarmStart), betaine (ranging 0.8, 1, 1.2, 1.4 or 1.6 M), supplementary MgSO4 (ranging 2, 4, 6 or 8 mM) and 8 U of the tested Bst polymerase in each case with 2 μL of template DNA. .. All LAMP reactions mixtures were performed in 0.5-mL micro centrifuge tubes that were incubated in a heating block (K Dry-Bath) at a range of temperatures (61, 63 and 65°C) for 60 min to optimize the reaction conditions and then heated at 80°C for 5–10 min to inactivate the enzyme and thus to terminate the reaction.

    SYBR Green Assay:

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    Article Title: Rapid and sensitive detection of Pseudocercospora eumusae pathogen causing eumusae leaf spot disease of banana by loop-mediated isothermal amplification (LAMP) method
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    Incubation:

    Article Title: Strong-LAMP: A LAMP Assay for Strongyloides spp. Detection in Stool and Urine Samples. Towards the Diagnosis of Human Strongyloidiasis Starting from a Rodent Model
    Article Snippet: Thus, LAMP reactions mixtures (25 μL) contained 40 pmol of each FIP and BIP primers, 5 pmol of each F3 and B3 primers, 1.4 mM of each dNTP (Bioron), 1x ThermoPol Reaction Buffer -20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4 )2 SO4 , 2 mM MgSO4 , 0.1% Triton X-100; New England Biolabs, UK- (when using Bst Polymerase Large Fragment) or 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- (when using either Bst DNA polymerase 2.0 or Bst DNA polymerase 2.0 WarmStart), betaine (ranging 0.8, 1, 1.2, 1.4 or 1.6 M), supplementary MgSO4 (ranging 2, 4, 6 or 8 mM) and 8 U of the tested Bst polymerase in each case with 2 μL of template DNA. .. All LAMP reactions mixtures were performed in 0.5-mL micro centrifuge tubes that were incubated in a heating block (K Dry-Bath) at a range of temperatures (61, 63 and 65°C) for 60 min to optimize the reaction conditions and then heated at 80°C for 5–10 min to inactivate the enzyme and thus to terminate the reaction.

    Article Title: Charged-to-Alanine Scanning Mutagenesis of the N-Terminal Half of Adeno-Associated Virus Type 2 Rep78 Protein
    Article Snippet: The samples were mixed with 20 μg of proteinase K, incubated for 6 h at 55°C, extracted with an equal volume of phenol-chloroform, ethanol precipitated, and then suspended in 200 μl of TE. .. One microliter of isolated genomic DNA was subjected to a thermal cycling reaction in a 20-μl reaction mixture containing 1× thermophilic DNA polymerase buffer [10 mM KCl, 20 mM Tris-HCl (pH 8.8), 10 mM (NH4 )2 SO4 , 4 mM MgSO4 , 0.1% Triton X-100 (New England Biolabs {NEB})], 1 μM 5′-CGGCCTCAGTGAGCGAGCGAGC and 5′-CGGGGAGGATCCGCTCAGAGGACA, and 2 U of Deep Vent Exo(−) DNA polymerase (NEB).

    Article Title: Rapid and sensitive detection of Pseudocercospora eumusae pathogen causing eumusae leaf spot disease of banana by loop-mediated isothermal amplification (LAMP) method
    Article Snippet: .. To determine the optimal reaction temperature and time, the LAMP reaction mixtures were incubated at 56–65 °C for 15, 30, 45, 60, and 90 min. A total volume of 25 ul for the LAMP reaction contained 0.48 µM FIP and BIP, 0.12 µM concentrations of F3 and B3, 2 mM dNTPs mix, 2.0 U of Bst DNA polymerase large fragment (New England Biolabs, Inc., Beverly, MA), 1× ThermoPol buffer (20 mM Tris–HCl, 10 mM (NH4 )2 SO4 , 10 mM KCl, 2 mM MgSO4 , 0.1% Triton X-100, pH 8.8) (New England Biolabs), 4 mM MgSO4 and 1 µl of template DNA (25 ng). .. The visual detection of LAMP products was performed by the addition of 2 µl of 1000× SYBR® Green I nucleic acid gel stain (Invitrogen, Carlsbad, California).

    Article Title: A general strategy for expanding polymerase function by droplet microfluidics
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    Gel Extraction:

    Article Title: Improved nucleotide selectivity and termination of 3?-OH unblocked reversible terminators by molecular tuning of 2-nitrobenzyl alkylated HOMedU triphosphates
    Article Snippet: Approximately 50 ng of genomic DNA was amplified with 0.4 µM of HNF1a_2.1 (F/R) or HNF1a_10.1 (F/R) primer pairs and one unit of Vent(exo− ) polymerase in 1× ThermoPol buffer [20 mM Tris–HCl, pH 8.8; 10 mM (NH4 )2 SO4 ; 10 mM KCl; 2 mM MgSO4 ; 0.1% Triton X-100; New England BioLabs], 1 M betaine ( , ) and 200 μM each of dATP, dCTP, dGTP and either TTP or HOMedUTP. .. Cycling conditions were 4 min at 95°C, then 35 cycles of 95°C for 30 s, 59°C for 30 s and 72°C for 90 s with a final step at 72°C for seven min. PCR products were purified using a Qiagen QIAquick gel extraction kit and sequenced using a ‘1 /16 ’ dilution of BigDye version 3.1 chemistry.

    Activity Assay:

    Article Title: A general strategy for expanding polymerase function by droplet microfluidics
    Article Snippet: Paragraph title: Polymerase activity assays ... The primer–template complex was annealed in ThermoPol buffer (1 × : 20 mM Tris-HCl, 10 mM (NH4)2SO4, 10 mM KCl, 2 mM MgSO4, 0.1% Triton X-100, pH 8.8; New England Biolabs) by heating for 5 min at 95 °C and cooling for 5 min at 4 °C.

    Expressing:

    Article Title: Charged-to-Alanine Scanning Mutagenesis of the N-Terminal Half of Adeno-Associated Virus Type 2 Rep78 Protein
    Article Snippet: Two micrograms of pCMVR78, mutant Rep expression plasmid, or blank vector was transfected into 2 × 105 293 cells/well in six-well plates along with 2 μg of pW1, harboring a lacZ expression cassette flanked by ITRs, by a standard calcium phosphate precipitation method. .. One microliter of isolated genomic DNA was subjected to a thermal cycling reaction in a 20-μl reaction mixture containing 1× thermophilic DNA polymerase buffer [10 mM KCl, 20 mM Tris-HCl (pH 8.8), 10 mM (NH4 )2 SO4 , 4 mM MgSO4 , 0.1% Triton X-100 (New England Biolabs {NEB})], 1 μM 5′-CGGCCTCAGTGAGCGAGCGAGC and 5′-CGGGGAGGATCCGCTCAGAGGACA, and 2 U of Deep Vent Exo(−) DNA polymerase (NEB).

    Modification:

    Article Title: Evaluating the influence of life‐history characteristics on genetic structure: a comparison of small mammals inhabiting complex agricultural landscapes
    Article Snippet: Microsatellite genotyping We used an ammonium acetate protocol and an ethanol wash to extract DNA from tissue samples (modified from the PUREGENE kit; Gentra Systems, Minneapolis, MN). .. Amplification by multiplex PCR took place in 10 μ L volumes with 20 ng of template DNA, 0.2 mM of each dNTP, 1 U of Taq DNA polymerase (NEB), and 2× Thermopol reaction buffer (20 mM Tris‐HCl, 10 mM (NH4 )2 SO4 , 10 mM KCl, 2 mM MgSO4 , 0.1% Triton X‐100; NEB).

    Article Title: Mutational Disruption of cis-Acting Replication Element 2C in Coxsackievirus B3 Leads to 5′-Terminal Genomic Deletions
    Article Snippet: Cycling was carried out as noted in with modified cycle denaturation and annealing times of 30 s each. .. The two amplimers were gel purified, combined in Thermopol reaction buffer [20 mM Tris-HCl, 10 mM (NH4 )2 SO4 , 10 mM KCl, 4 mM MgSO4 , 0.1% Triton X-100, pH 8.8; New England BioLabs] with 200 μM dNTPs, and repeatedly denatured and annealed 10 times (1 min at 94°C, 50°C for 1 min), followed by extension with Deep Vent polymerase (2,000 U/ml) at 72°C for 2 min.

    Derivative Assay:

    Article Title: Rapid and sensitive detection of Pseudocercospora eumusae pathogen causing eumusae leaf spot disease of banana by loop-mediated isothermal amplification (LAMP) method
    Article Snippet: The LAMP primers specific to P. eumusae were designed based on the specific SCAR marker sequence of P. eumusae derived from RAPD analysis. .. To determine the optimal reaction temperature and time, the LAMP reaction mixtures were incubated at 56–65 °C for 15, 30, 45, 60, and 90 min. A total volume of 25 ul for the LAMP reaction contained 0.48 µM FIP and BIP, 0.12 µM concentrations of F3 and B3, 2 mM dNTPs mix, 2.0 U of Bst DNA polymerase large fragment (New England Biolabs, Inc., Beverly, MA), 1× ThermoPol buffer (20 mM Tris–HCl, 10 mM (NH4 )2 SO4 , 10 mM KCl, 2 mM MgSO4 , 0.1% Triton X-100, pH 8.8) (New England Biolabs), 4 mM MgSO4 and 1 µl of template DNA (25 ng).

    Hybridization:

    Article Title: Charged-to-Alanine Scanning Mutagenesis of the N-Terminal Half of Adeno-Associated Virus Type 2 Rep78 Protein
    Article Snippet: One microliter of isolated genomic DNA was subjected to a thermal cycling reaction in a 20-μl reaction mixture containing 1× thermophilic DNA polymerase buffer [10 mM KCl, 20 mM Tris-HCl (pH 8.8), 10 mM (NH4 )2 SO4 , 4 mM MgSO4 , 0.1% Triton X-100 (New England Biolabs {NEB})], 1 μM 5′-CGGCCTCAGTGAGCGAGCGAGC and 5′-CGGGGAGGATCCGCTCAGAGGACA, and 2 U of Deep Vent Exo(−) DNA polymerase (NEB). .. Ten microliters of the PCR mixture was transferred to a hybridization membrane (Hybond-N+; Amersham) by using a dot blot apparatus and hybridized with an AAVS1 probe.

    High Performance Liquid Chromatography:

    Article Title: Improved nucleotide selectivity and termination of 3?-OH unblocked reversible terminators by molecular tuning of 2-nitrobenzyl alkylated HOMedU triphosphates
    Article Snippet: Approximately 50 ng of genomic DNA was amplified with 0.4 µM of HNF1a_2.1 (F/R) or HNF1a_10.1 (F/R) primer pairs and one unit of Vent(exo− ) polymerase in 1× ThermoPol buffer [20 mM Tris–HCl, pH 8.8; 10 mM (NH4 )2 SO4 ; 10 mM KCl; 2 mM MgSO4 ; 0.1% Triton X-100; New England BioLabs], 1 M betaine ( , ) and 200 μM each of dATP, dCTP, dGTP and either TTP or HOMedUTP. .. Reaction products were purified by ethanol precipitation, resuspended in 30 µl of HPLC water, and then loaded onto an AB model 3100 DNA sequencer.

    Transfection:

    Article Title: Charged-to-Alanine Scanning Mutagenesis of the N-Terminal Half of Adeno-Associated Virus Type 2 Rep78 Protein
    Article Snippet: Two micrograms of pCMVR78, mutant Rep expression plasmid, or blank vector was transfected into 2 × 105 293 cells/well in six-well plates along with 2 μg of pW1, harboring a lacZ expression cassette flanked by ITRs, by a standard calcium phosphate precipitation method. .. One microliter of isolated genomic DNA was subjected to a thermal cycling reaction in a 20-μl reaction mixture containing 1× thermophilic DNA polymerase buffer [10 mM KCl, 20 mM Tris-HCl (pH 8.8), 10 mM (NH4 )2 SO4 , 4 mM MgSO4 , 0.1% Triton X-100 (New England Biolabs {NEB})], 1 μM 5′-CGGCCTCAGTGAGCGAGCGAGC and 5′-CGGGGAGGATCCGCTCAGAGGACA, and 2 U of Deep Vent Exo(−) DNA polymerase (NEB).

    Generated:

    Article Title: Charged-to-Alanine Scanning Mutagenesis of the N-Terminal Half of Adeno-Associated Virus Type 2 Rep78 Protein
    Article Snippet: One microliter of isolated genomic DNA was subjected to a thermal cycling reaction in a 20-μl reaction mixture containing 1× thermophilic DNA polymerase buffer [10 mM KCl, 20 mM Tris-HCl (pH 8.8), 10 mM (NH4 )2 SO4 , 4 mM MgSO4 , 0.1% Triton X-100 (New England Biolabs {NEB})], 1 μM 5′-CGGCCTCAGTGAGCGAGCGAGC and 5′-CGGGGAGGATCCGCTCAGAGGACA, and 2 U of Deep Vent Exo(−) DNA polymerase (NEB). .. The probe was a random-primed 32 P-labeled PCR fragment generated in a reaction with 20 ng of pRVK as a template and primers 5′-ACTTTGAGCTCTACTGGCTTC and 5′-GGAGGATCCGCTCAGAGG.

    Article Title: Mutational Disruption of cis-Acting Replication Element 2C in Coxsackievirus B3 Leads to 5′-Terminal Genomic Deletions
    Article Snippet: Two overlapping fragments of CVB3 cDNA were generated from the infectious cDNA clone of CVB3/28 ( ) by PCR using Deep Vent DNA polymerase (New England BioLabs, Ipswich, MA) and the primer pair ID13 and CREKORev or CREKO and XBA ( ) with 4 mM MgSO4 and 200 μM deoxynucleoside triphosphates (dNTPs). .. The two amplimers were gel purified, combined in Thermopol reaction buffer [20 mM Tris-HCl, 10 mM (NH4 )2 SO4 , 10 mM KCl, 4 mM MgSO4 , 0.1% Triton X-100, pH 8.8; New England BioLabs] with 200 μM dNTPs, and repeatedly denatured and annealed 10 times (1 min at 94°C, 50°C for 1 min), followed by extension with Deep Vent polymerase (2,000 U/ml) at 72°C for 2 min.

    Imaging:

    Article Title: Charged-to-Alanine Scanning Mutagenesis of the N-Terminal Half of Adeno-Associated Virus Type 2 Rep78 Protein
    Article Snippet: One microliter of isolated genomic DNA was subjected to a thermal cycling reaction in a 20-μl reaction mixture containing 1× thermophilic DNA polymerase buffer [10 mM KCl, 20 mM Tris-HCl (pH 8.8), 10 mM (NH4 )2 SO4 , 4 mM MgSO4 , 0.1% Triton X-100 (New England Biolabs {NEB})], 1 μM 5′-CGGCCTCAGTGAGCGAGCGAGC and 5′-CGGGGAGGATCCGCTCAGAGGACA, and 2 U of Deep Vent Exo(−) DNA polymerase (NEB). .. The membranes were then analyzed on a BAS-1500 imaging analyzer.

    Isolation:

    Article Title: Discerning non-disjunction in Down syndrome patients by means of GluK1-(AGAT)n and D21S2055-(GATA)n microsatellites on chromosome 21
    Article Snippet: Genotyping procedures Genomic DNA was isolated from whole blood lymphocytes by the salting-out procedure. .. For the GluK1-(AGAT)n polymorphism, 5 pmol of each forward (5’-GCTAAATAGATATATGATAAACGG-3’) and reverse (5’-CTGGCAGTAAATGTCTATGAAAC-3’) primers[ ] were used in reactions containing 100 ng of template DNA, 1-X Thermopol-II buffer (NEB) containing of 10 mMKCl, 10 mM (NH4 )2 SO4, 20 mMTris-HCl (pH 8.8 at 25°C), 0.1% Triton X-100 (NEB), 1 mM MgSO4 , 200 μM dNTPs, and 0.2 U Taq DNA polymerase in 20μl reaction volume.

    Article Title: Charged-to-Alanine Scanning Mutagenesis of the N-Terminal Half of Adeno-Associated Virus Type 2 Rep78 Protein
    Article Snippet: .. One microliter of isolated genomic DNA was subjected to a thermal cycling reaction in a 20-μl reaction mixture containing 1× thermophilic DNA polymerase buffer [10 mM KCl, 20 mM Tris-HCl (pH 8.8), 10 mM (NH4 )2 SO4 , 4 mM MgSO4 , 0.1% Triton X-100 (New England Biolabs {NEB})], 1 μM 5′-CGGCCTCAGTGAGCGAGCGAGC and 5′-CGGGGAGGATCCGCTCAGAGGACA, and 2 U of Deep Vent Exo(−) DNA polymerase (NEB). .. Ten microliters of the PCR mixture was transferred to a hybridization membrane (Hybond-N+; Amersham) by using a dot blot apparatus and hybridized with an AAVS1 probe.

    Sequencing:

    Article Title: In vitro selection of an XNA aptamer capable of small-molecule recognition
    Article Snippet: A single stranded DNA library having the sequence 5′-GCGCATACCAGCTTATTCAATT-N50 -AGATAGTAAGTGCAATCTCGGC-3′ (20 pmol) was amplified in 50 μl PCR reactions containing 0.2 μM template, 0.2 μM each primers, 200 μM dNTPs, and 2.5 U Taq DNA polymerase in 1× Thermopol buffer (20 mM Tris–HCl, 10 mM (NH4 )2 SO4 , 10 mM KCl. .. 2 mM MgSO4 , 0.1% Triton X-100, pH 8.8, New England Biolabs).

    Article Title: Improved nucleotide selectivity and termination of 3?-OH unblocked reversible terminators by molecular tuning of 2-nitrobenzyl alkylated HOMedU triphosphates
    Article Snippet: Paragraph title: PCR and Sanger sequencing ... Approximately 50 ng of genomic DNA was amplified with 0.4 µM of HNF1a_2.1 (F/R) or HNF1a_10.1 (F/R) primer pairs and one unit of Vent(exo− ) polymerase in 1× ThermoPol buffer [20 mM Tris–HCl, pH 8.8; 10 mM (NH4 )2 SO4 ; 10 mM KCl; 2 mM MgSO4 ; 0.1% Triton X-100; New England BioLabs], 1 M betaine ( , ) and 200 μM each of dATP, dCTP, dGTP and either TTP or HOMedUTP.

    Article Title: Rapid and sensitive detection of Pseudocercospora eumusae pathogen causing eumusae leaf spot disease of banana by loop-mediated isothermal amplification (LAMP) method
    Article Snippet: The LAMP primers specific to P. eumusae were designed based on the specific SCAR marker sequence of P. eumusae derived from RAPD analysis. .. To determine the optimal reaction temperature and time, the LAMP reaction mixtures were incubated at 56–65 °C for 15, 30, 45, 60, and 90 min. A total volume of 25 ul for the LAMP reaction contained 0.48 µM FIP and BIP, 0.12 µM concentrations of F3 and B3, 2 mM dNTPs mix, 2.0 U of Bst DNA polymerase large fragment (New England Biolabs, Inc., Beverly, MA), 1× ThermoPol buffer (20 mM Tris–HCl, 10 mM (NH4 )2 SO4 , 10 mM KCl, 2 mM MgSO4 , 0.1% Triton X-100, pH 8.8) (New England Biolabs), 4 mM MgSO4 and 1 µl of template DNA (25 ng).

    Fluorescence:

    Article Title: A general strategy for expanding polymerase function by droplet microfluidics
    Article Snippet: Primer-extension reactions were analysed by denaturing PAGE or fluorescence. .. The primer–template complex was annealed in ThermoPol buffer (1 × : 20 mM Tris-HCl, 10 mM (NH4)2SO4, 10 mM KCl, 2 mM MgSO4, 0.1% Triton X-100, pH 8.8; New England Biolabs) by heating for 5 min at 95 °C and cooling for 5 min at 4 °C.

    Article Title: Development and application of a rapid and visual loop-mediated isothermal amplification for the detection of Sporisorium scitamineum in sugarcane
    Article Snippet: LAMP products of combination 7 showed high flavogreen fluorescence, and displayed bright typical ladder-like bands after electrophoresis, apparent in distinction from the negative and blank controls, whereas combinations 4, 8 and 10 showed intensive flavogreen fluorescent signals but weaker band brightness than combination 7 in electrophoresis. .. The optimized LAMP reaction conditions including 4 mmol/L MgSO4 (50 mmol/L), 1.4 mmol/L dNTPs (10 mmol/L), 2.5 μL 10×ThermoPol Reaction Buffer [containing 20 mmol/L Tris-HCl pH 8.8, 10 mmol/L KCl, 2 mmol/L MgSO4 , 10 mmol/L (NH4 )2 SO4 , 0.1% Triton X-100, NEB company], 1.2 μmol/L each of FIP and BIP primers, 0.2 μmol/L each of F3 and B3 primers, 8.0 U Bst DNA polymerase (8 000 U/mL, NEB company), DNA template 1.0 μL, ddH2 O were complemented to a volume of 25 μL.

    Mutagenesis:

    Article Title: Charged-to-Alanine Scanning Mutagenesis of the N-Terminal Half of Adeno-Associated Virus Type 2 Rep78 Protein
    Article Snippet: Two micrograms of pCMVR78, mutant Rep expression plasmid, or blank vector was transfected into 2 × 105 293 cells/well in six-well plates along with 2 μg of pW1, harboring a lacZ expression cassette flanked by ITRs, by a standard calcium phosphate precipitation method. .. One microliter of isolated genomic DNA was subjected to a thermal cycling reaction in a 20-μl reaction mixture containing 1× thermophilic DNA polymerase buffer [10 mM KCl, 20 mM Tris-HCl (pH 8.8), 10 mM (NH4 )2 SO4 , 4 mM MgSO4 , 0.1% Triton X-100 (New England Biolabs {NEB})], 1 μM 5′-CGGCCTCAGTGAGCGAGCGAGC and 5′-CGGGGAGGATCCGCTCAGAGGACA, and 2 U of Deep Vent Exo(−) DNA polymerase (NEB).

    Article Title: Mutational Disruption of cis-Acting Replication Element 2C in Coxsackievirus B3 Leads to 5′-Terminal Genomic Deletions
    Article Snippet: The mutations in the CVB3 CRE(2C) were generated using overlap extension mutagenesis ( ). .. The two amplimers were gel purified, combined in Thermopol reaction buffer [20 mM Tris-HCl, 10 mM (NH4 )2 SO4 , 10 mM KCl, 4 mM MgSO4 , 0.1% Triton X-100, pH 8.8; New England BioLabs] with 200 μM dNTPs, and repeatedly denatured and annealed 10 times (1 min at 94°C, 50°C for 1 min), followed by extension with Deep Vent polymerase (2,000 U/ml) at 72°C for 2 min.

    Random Primed:

    Article Title: Charged-to-Alanine Scanning Mutagenesis of the N-Terminal Half of Adeno-Associated Virus Type 2 Rep78 Protein
    Article Snippet: One microliter of isolated genomic DNA was subjected to a thermal cycling reaction in a 20-μl reaction mixture containing 1× thermophilic DNA polymerase buffer [10 mM KCl, 20 mM Tris-HCl (pH 8.8), 10 mM (NH4 )2 SO4 , 4 mM MgSO4 , 0.1% Triton X-100 (New England Biolabs {NEB})], 1 μM 5′-CGGCCTCAGTGAGCGAGCGAGC and 5′-CGGGGAGGATCCGCTCAGAGGACA, and 2 U of Deep Vent Exo(−) DNA polymerase (NEB). .. The probe was a random-primed 32 P-labeled PCR fragment generated in a reaction with 20 ng of pRVK as a template and primers 5′-ACTTTGAGCTCTACTGGCTTC and 5′-GGAGGATCCGCTCAGAGG.

    Negative Control:

    Article Title: Strong-LAMP: A LAMP Assay for Strongyloides spp. Detection in Stool and Urine Samples. Towards the Diagnosis of Human Strongyloidiasis Starting from a Rodent Model
    Article Snippet: Thus, LAMP reactions mixtures (25 μL) contained 40 pmol of each FIP and BIP primers, 5 pmol of each F3 and B3 primers, 1.4 mM of each dNTP (Bioron), 1x ThermoPol Reaction Buffer -20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4 )2 SO4 , 2 mM MgSO4 , 0.1% Triton X-100; New England Biolabs, UK- (when using Bst Polymerase Large Fragment) or 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- (when using either Bst DNA polymerase 2.0 or Bst DNA polymerase 2.0 WarmStart), betaine (ranging 0.8, 1, 1.2, 1.4 or 1.6 M), supplementary MgSO4 (ranging 2, 4, 6 or 8 mM) and 8 U of the tested Bst polymerase in each case with 2 μL of template DNA. .. Template DNA was replaced by ultrapure water as negative control in each LAMP reaction.

    Labeling:

    Article Title: In vitro selection of an XNA aptamer capable of small-molecule recognition
    Article Snippet: 2 mM MgSO4 , 0.1% Triton X-100, pH 8.8, New England Biolabs). .. The amplified double stranded DNA was purified using a PCR cleanup column (Qiagen) and the PEG-functionalized strand was separated from the FAM labeled strand on a denaturing 10% polyacrylamide gel.

    Mouse Assay:

    Article Title: Evaluating the influence of life‐history characteristics on genetic structure: a comparison of small mammals inhabiting complex agricultural landscapes
    Article Snippet: DNA quality was checked by running 3 μ L of DNA out on a 2% agarose gel stained with ethidium 12 bromide, and then, DNA extracts were diluted to a final concentration of approximately 10 ng/μ L. Chipmunk samples were amplified at 12 (EACH01‐12; Anderson et al. ) microsatellite loci, while white‐footed mice were amplified at 10 loci [PO‐26, PO‐85, Po‐97 (Prince et al. ); Pml01, Pml02, Pml05, Pml09, Pml12 (Chirhart et al. ); PLGT15 (Schmidt )]. .. Amplification by multiplex PCR took place in 10 μ L volumes with 20 ng of template DNA, 0.2 mM of each dNTP, 1 U of Taq DNA polymerase (NEB), and 2× Thermopol reaction buffer (20 mM Tris‐HCl, 10 mM (NH4 )2 SO4 , 10 mM KCl, 2 mM MgSO4 , 0.1% Triton X‐100; NEB).

    Dot Blot:

    Article Title: Charged-to-Alanine Scanning Mutagenesis of the N-Terminal Half of Adeno-Associated Virus Type 2 Rep78 Protein
    Article Snippet: One microliter of isolated genomic DNA was subjected to a thermal cycling reaction in a 20-μl reaction mixture containing 1× thermophilic DNA polymerase buffer [10 mM KCl, 20 mM Tris-HCl (pH 8.8), 10 mM (NH4 )2 SO4 , 4 mM MgSO4 , 0.1% Triton X-100 (New England Biolabs {NEB})], 1 μM 5′-CGGCCTCAGTGAGCGAGCGAGC and 5′-CGGGGAGGATCCGCTCAGAGGACA, and 2 U of Deep Vent Exo(−) DNA polymerase (NEB). .. Ten microliters of the PCR mixture was transferred to a hybridization membrane (Hybond-N+; Amersham) by using a dot blot apparatus and hybridized with an AAVS1 probe.

    Polymerase Chain Reaction:

    Article Title: Evaluating the influence of life‐history characteristics on genetic structure: a comparison of small mammals inhabiting complex agricultural landscapes
    Article Snippet: .. Amplification by multiplex PCR took place in 10 μ L volumes with 20 ng of template DNA, 0.2 mM of each dNTP, 1 U of Taq DNA polymerase (NEB), and 2× Thermopol reaction buffer (20 mM Tris‐HCl, 10 mM (NH4 )2 SO4 , 10 mM KCl, 2 mM MgSO4 , 0.1% Triton X‐100; NEB). .. The amount of primer for each locus (0.05–0.30 μ M) was adjusted so that all loci in the multiplex reaction would result in approximately equal intensities of product.

    Article Title: In vitro selection of an XNA aptamer capable of small-molecule recognition
    Article Snippet: Paragraph title: PCR amplification of ssDNA library ... 2 mM MgSO4 , 0.1% Triton X-100, pH 8.8, New England Biolabs).

    Article Title: Improved nucleotide selectivity and termination of 3?-OH unblocked reversible terminators by molecular tuning of 2-nitrobenzyl alkylated HOMedU triphosphates
    Article Snippet: Paragraph title: PCR and Sanger sequencing ... Approximately 50 ng of genomic DNA was amplified with 0.4 µM of HNF1a_2.1 (F/R) or HNF1a_10.1 (F/R) primer pairs and one unit of Vent(exo− ) polymerase in 1× ThermoPol buffer [20 mM Tris–HCl, pH 8.8; 10 mM (NH4 )2 SO4 ; 10 mM KCl; 2 mM MgSO4 ; 0.1% Triton X-100; New England BioLabs], 1 M betaine ( , ) and 200 μM each of dATP, dCTP, dGTP and either TTP or HOMedUTP.

    Article Title: Discerning non-disjunction in Down syndrome patients by means of GluK1-(AGAT)n and D21S2055-(GATA)n microsatellites on chromosome 21
    Article Snippet: [ ] PCR-based amplification of genomic DNA targets was carried out in the DNA Engine Thermal Cycler (MJ Research PTC-200). .. For the GluK1-(AGAT)n polymorphism, 5 pmol of each forward (5’-GCTAAATAGATATATGATAAACGG-3’) and reverse (5’-CTGGCAGTAAATGTCTATGAAAC-3’) primers[ ] were used in reactions containing 100 ng of template DNA, 1-X Thermopol-II buffer (NEB) containing of 10 mMKCl, 10 mM (NH4 )2 SO4, 20 mMTris-HCl (pH 8.8 at 25°C), 0.1% Triton X-100 (NEB), 1 mM MgSO4 , 200 μM dNTPs, and 0.2 U Taq DNA polymerase in 20μl reaction volume.

    Article Title: Charged-to-Alanine Scanning Mutagenesis of the N-Terminal Half of Adeno-Associated Virus Type 2 Rep78 Protein
    Article Snippet: Paragraph title: PCR-based assay for site-specific integration at the AAVS1 locus. ... One microliter of isolated genomic DNA was subjected to a thermal cycling reaction in a 20-μl reaction mixture containing 1× thermophilic DNA polymerase buffer [10 mM KCl, 20 mM Tris-HCl (pH 8.8), 10 mM (NH4 )2 SO4 , 4 mM MgSO4 , 0.1% Triton X-100 (New England Biolabs {NEB})], 1 μM 5′-CGGCCTCAGTGAGCGAGCGAGC and 5′-CGGGGAGGATCCGCTCAGAGGACA, and 2 U of Deep Vent Exo(−) DNA polymerase (NEB).

    Article Title: Mutational Disruption of cis-Acting Replication Element 2C in Coxsackievirus B3 Leads to 5′-Terminal Genomic Deletions
    Article Snippet: Paragraph title: Mutational disruption of CRE(2C) using overlap extension PCR. ... The two amplimers were gel purified, combined in Thermopol reaction buffer [20 mM Tris-HCl, 10 mM (NH4 )2 SO4 , 10 mM KCl, 4 mM MgSO4 , 0.1% Triton X-100, pH 8.8; New England BioLabs] with 200 μM dNTPs, and repeatedly denatured and annealed 10 times (1 min at 94°C, 50°C for 1 min), followed by extension with Deep Vent polymerase (2,000 U/ml) at 72°C for 2 min.

    Polyacrylamide Gel Electrophoresis:

    Article Title: A general strategy for expanding polymerase function by droplet microfluidics
    Article Snippet: Primer-extension reactions were analysed by denaturing PAGE or fluorescence. .. The primer–template complex was annealed in ThermoPol buffer (1 × : 20 mM Tris-HCl, 10 mM (NH4)2SO4, 10 mM KCl, 2 mM MgSO4, 0.1% Triton X-100, pH 8.8; New England Biolabs) by heating for 5 min at 95 °C and cooling for 5 min at 4 °C.

    Salting Out:

    Article Title: Discerning non-disjunction in Down syndrome patients by means of GluK1-(AGAT)n and D21S2055-(GATA)n microsatellites on chromosome 21
    Article Snippet: Genotyping procedures Genomic DNA was isolated from whole blood lymphocytes by the salting-out procedure. .. For the GluK1-(AGAT)n polymorphism, 5 pmol of each forward (5’-GCTAAATAGATATATGATAAACGG-3’) and reverse (5’-CTGGCAGTAAATGTCTATGAAAC-3’) primers[ ] were used in reactions containing 100 ng of template DNA, 1-X Thermopol-II buffer (NEB) containing of 10 mMKCl, 10 mM (NH4 )2 SO4, 20 mMTris-HCl (pH 8.8 at 25°C), 0.1% Triton X-100 (NEB), 1 mM MgSO4 , 200 μM dNTPs, and 0.2 U Taq DNA polymerase in 20μl reaction volume.

    Purification:

    Article Title: In vitro selection of an XNA aptamer capable of small-molecule recognition
    Article Snippet: 2 mM MgSO4 , 0.1% Triton X-100, pH 8.8, New England Biolabs). .. The amplified double stranded DNA was purified using a PCR cleanup column (Qiagen) and the PEG-functionalized strand was separated from the FAM labeled strand on a denaturing 10% polyacrylamide gel.

    Article Title: Improved nucleotide selectivity and termination of 3?-OH unblocked reversible terminators by molecular tuning of 2-nitrobenzyl alkylated HOMedU triphosphates
    Article Snippet: Approximately 50 ng of genomic DNA was amplified with 0.4 µM of HNF1a_2.1 (F/R) or HNF1a_10.1 (F/R) primer pairs and one unit of Vent(exo− ) polymerase in 1× ThermoPol buffer [20 mM Tris–HCl, pH 8.8; 10 mM (NH4 )2 SO4 ; 10 mM KCl; 2 mM MgSO4 ; 0.1% Triton X-100; New England BioLabs], 1 M betaine ( , ) and 200 μM each of dATP, dCTP, dGTP and either TTP or HOMedUTP. .. Cycling conditions were 4 min at 95°C, then 35 cycles of 95°C for 30 s, 59°C for 30 s and 72°C for 90 s with a final step at 72°C for seven min. PCR products were purified using a Qiagen QIAquick gel extraction kit and sequenced using a ‘1 /16 ’ dilution of BigDye version 3.1 chemistry.

    Article Title: Mutational Disruption of cis-Acting Replication Element 2C in Coxsackievirus B3 Leads to 5′-Terminal Genomic Deletions
    Article Snippet: .. The two amplimers were gel purified, combined in Thermopol reaction buffer [20 mM Tris-HCl, 10 mM (NH4 )2 SO4 , 10 mM KCl, 4 mM MgSO4 , 0.1% Triton X-100, pH 8.8; New England BioLabs] with 200 μM dNTPs, and repeatedly denatured and annealed 10 times (1 min at 94°C, 50°C for 1 min), followed by extension with Deep Vent polymerase (2,000 U/ml) at 72°C for 2 min. .. The mutated overlap extension product was subsequently directly amplified using the ID13 and XBA primers ( and ) and Deep Vent polymerase with the modified denaturation and annealing times described above.

    Plasmid Preparation:

    Article Title: Novel thermostable Y-family polymerases: applications for the PCR amplification of damaged or ancient DNAs
    Article Snippet: .. PCRs on undamaged DNA For the undamaged plasmid DNA template, 50 µl PCRs were set up containing standard ThermoPol reaction buffer [20 mM Tris–HCl, pH 8.8, 10 mM KCl, 10 mM (NH4 )2 SO4 , 2 mM MgSO4 , 0.1% Triton X-100] (New England Biolabs), 10 ng of circular pJM548 (Ste dpo4 -like in pGEM-T), 200 µM of each ultrapure dNTP and 1 µM of each primer; Ste_FNde5 and Ste_RBam1089 whose sequences are provided above. .. Aliquots containing 2.5 U of Taq DNA polymerase (New England Biolabs) were used as a positive control.

    Article Title: Charged-to-Alanine Scanning Mutagenesis of the N-Terminal Half of Adeno-Associated Virus Type 2 Rep78 Protein
    Article Snippet: Two micrograms of pCMVR78, mutant Rep expression plasmid, or blank vector was transfected into 2 × 105 293 cells/well in six-well plates along with 2 μg of pW1, harboring a lacZ expression cassette flanked by ITRs, by a standard calcium phosphate precipitation method. .. One microliter of isolated genomic DNA was subjected to a thermal cycling reaction in a 20-μl reaction mixture containing 1× thermophilic DNA polymerase buffer [10 mM KCl, 20 mM Tris-HCl (pH 8.8), 10 mM (NH4 )2 SO4 , 4 mM MgSO4 , 0.1% Triton X-100 (New England Biolabs {NEB})], 1 μM 5′-CGGCCTCAGTGAGCGAGCGAGC and 5′-CGGGGAGGATCCGCTCAGAGGACA, and 2 U of Deep Vent Exo(−) DNA polymerase (NEB).

    Software:

    Article Title: Evaluating the influence of life‐history characteristics on genetic structure: a comparison of small mammals inhabiting complex agricultural landscapes
    Article Snippet: Amplification by multiplex PCR took place in 10 μ L volumes with 20 ng of template DNA, 0.2 mM of each dNTP, 1 U of Taq DNA polymerase (NEB), and 2× Thermopol reaction buffer (20 mM Tris‐HCl, 10 mM (NH4 )2 SO4 , 10 mM KCl, 2 mM MgSO4 , 0.1% Triton X‐100; NEB). .. The PCR products were sized on an Applied Biosystems 3730 automated sequencer, and the genotypes were determined for all loci in all individuals using the software GeneMapper 3.7 (Applied Biosystems, Foster City, CA).

    Electrophoresis:

    Article Title: Development and application of a rapid and visual loop-mediated isothermal amplification for the detection of Sporisorium scitamineum in sugarcane
    Article Snippet: LAMP products of combination 7 showed high flavogreen fluorescence, and displayed bright typical ladder-like bands after electrophoresis, apparent in distinction from the negative and blank controls, whereas combinations 4, 8 and 10 showed intensive flavogreen fluorescent signals but weaker band brightness than combination 7 in electrophoresis. .. The optimized LAMP reaction conditions including 4 mmol/L MgSO4 (50 mmol/L), 1.4 mmol/L dNTPs (10 mmol/L), 2.5 μL 10×ThermoPol Reaction Buffer [containing 20 mmol/L Tris-HCl pH 8.8, 10 mmol/L KCl, 2 mmol/L MgSO4 , 10 mmol/L (NH4 )2 SO4 , 0.1% Triton X-100, NEB company], 1.2 μmol/L each of FIP and BIP primers, 0.2 μmol/L each of F3 and B3 primers, 8.0 U Bst DNA polymerase (8 000 U/mL, NEB company), DNA template 1.0 μL, ddH2 O were complemented to a volume of 25 μL.

    Multiplex Assay:

    Article Title: Evaluating the influence of life‐history characteristics on genetic structure: a comparison of small mammals inhabiting complex agricultural landscapes
    Article Snippet: .. Amplification by multiplex PCR took place in 10 μ L volumes with 20 ng of template DNA, 0.2 mM of each dNTP, 1 U of Taq DNA polymerase (NEB), and 2× Thermopol reaction buffer (20 mM Tris‐HCl, 10 mM (NH4 )2 SO4 , 10 mM KCl, 2 mM MgSO4 , 0.1% Triton X‐100; NEB). .. The amount of primer for each locus (0.05–0.30 μ M) was adjusted so that all loci in the multiplex reaction would result in approximately equal intensities of product.

    Agarose Gel Electrophoresis:

    Article Title: Evaluating the influence of life‐history characteristics on genetic structure: a comparison of small mammals inhabiting complex agricultural landscapes
    Article Snippet: DNA quality was checked by running 3 μ L of DNA out on a 2% agarose gel stained with ethidium 12 bromide, and then, DNA extracts were diluted to a final concentration of approximately 10 ng/μ L. Chipmunk samples were amplified at 12 (EACH01‐12; Anderson et al. ) microsatellite loci, while white‐footed mice were amplified at 10 loci [PO‐26, PO‐85, Po‐97 (Prince et al. ); Pml01, Pml02, Pml05, Pml09, Pml12 (Chirhart et al. ); PLGT15 (Schmidt )]. .. Amplification by multiplex PCR took place in 10 μ L volumes with 20 ng of template DNA, 0.2 mM of each dNTP, 1 U of Taq DNA polymerase (NEB), and 2× Thermopol reaction buffer (20 mM Tris‐HCl, 10 mM (NH4 )2 SO4 , 10 mM KCl, 2 mM MgSO4 , 0.1% Triton X‐100; NEB).

    Article Title: Novel thermostable Y-family polymerases: applications for the PCR amplification of damaged or ancient DNAs
    Article Snippet: PCRs on undamaged DNA For the undamaged plasmid DNA template, 50 µl PCRs were set up containing standard ThermoPol reaction buffer [20 mM Tris–HCl, pH 8.8, 10 mM KCl, 10 mM (NH4 )2 SO4 , 2 mM MgSO4 , 0.1% Triton X-100] (New England Biolabs), 10 ng of circular pJM548 (Ste dpo4 -like in pGEM-T), 200 µM of each ultrapure dNTP and 1 µM of each primer; Ste_FNde5 and Ste_RBam1089 whose sequences are provided above. .. Reactions were heated to 85°C for 3 min and then treated with 35 cycles of 85°C for 30 s, 56°C for 1 min and 60°C for 4 min. Products were separated on a 0.9% agarose gel stained with ethidium bromide.

    Size-exclusion Chromatography:

    Article Title: Discerning non-disjunction in Down syndrome patients by means of GluK1-(AGAT)n and D21S2055-(GATA)n microsatellites on chromosome 21
    Article Snippet: For the GluK1-(AGAT)n polymorphism, 5 pmol of each forward (5’-GCTAAATAGATATATGATAAACGG-3’) and reverse (5’-CTGGCAGTAAATGTCTATGAAAC-3’) primers[ ] were used in reactions containing 100 ng of template DNA, 1-X Thermopol-II buffer (NEB) containing of 10 mMKCl, 10 mM (NH4 )2 SO4, 20 mMTris-HCl (pH 8.8 at 25°C), 0.1% Triton X-100 (NEB), 1 mM MgSO4 , 200 μM dNTPs, and 0.2 U Taq DNA polymerase in 20μl reaction volume. .. The cycling conditions were as follows: Denaturation at 95°C for 10 min followed by 30 cycles of denaturation at 95°C for 40 sec; annealing at 58°C for 40 sec; elongation at 72°C for 40 sec; final elongation step at 72°C for 7 min. Amplicons and size standards were resolved in 12% polyacrylamide gels containing 5% glycerol.

    Ethanol Precipitation:

    Article Title: Improved nucleotide selectivity and termination of 3?-OH unblocked reversible terminators by molecular tuning of 2-nitrobenzyl alkylated HOMedU triphosphates
    Article Snippet: Approximately 50 ng of genomic DNA was amplified with 0.4 µM of HNF1a_2.1 (F/R) or HNF1a_10.1 (F/R) primer pairs and one unit of Vent(exo− ) polymerase in 1× ThermoPol buffer [20 mM Tris–HCl, pH 8.8; 10 mM (NH4 )2 SO4 ; 10 mM KCl; 2 mM MgSO4 ; 0.1% Triton X-100; New England BioLabs], 1 M betaine ( , ) and 200 μM each of dATP, dCTP, dGTP and either TTP or HOMedUTP. .. Reaction products were purified by ethanol precipitation, resuspended in 30 µl of HPLC water, and then loaded onto an AB model 3100 DNA sequencer.

    Concentration Assay:

    Article Title: Strong-LAMP: A LAMP Assay for Strongyloides spp. Detection in Stool and Urine Samples. Towards the Diagnosis of Human Strongyloidiasis Starting from a Rodent Model
    Article Snippet: Optimization of LAMP assay We tried to evaluate the LAMP primer set designed by using different in house reaction mixtures each containing a different Bst polymerase (namely, Bst DNA polymerase Large Fragment, Bst DNA polymerase 2.0 and Bst DNA polymerase 2.0 WarmStart; New England Biolabs, UK) as well as varying concentration of betaine (Sigma, USA) and supplementary MgSO4 (New England Biolabs, UK) to compare results in S . venezuelensis DNA amplification. .. Thus, LAMP reactions mixtures (25 μL) contained 40 pmol of each FIP and BIP primers, 5 pmol of each F3 and B3 primers, 1.4 mM of each dNTP (Bioron), 1x ThermoPol Reaction Buffer -20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4 )2 SO4 , 2 mM MgSO4 , 0.1% Triton X-100; New England Biolabs, UK- (when using Bst Polymerase Large Fragment) or 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- (when using either Bst DNA polymerase 2.0 or Bst DNA polymerase 2.0 WarmStart), betaine (ranging 0.8, 1, 1.2, 1.4 or 1.6 M), supplementary MgSO4 (ranging 2, 4, 6 or 8 mM) and 8 U of the tested Bst polymerase in each case with 2 μL of template DNA.

    Article Title: Evaluating the influence of life‐history characteristics on genetic structure: a comparison of small mammals inhabiting complex agricultural landscapes
    Article Snippet: DNA quality was checked by running 3 μ L of DNA out on a 2% agarose gel stained with ethidium 12 bromide, and then, DNA extracts were diluted to a final concentration of approximately 10 ng/μ L. Chipmunk samples were amplified at 12 (EACH01‐12; Anderson et al. ) microsatellite loci, while white‐footed mice were amplified at 10 loci [PO‐26, PO‐85, Po‐97 (Prince et al. ); Pml01, Pml02, Pml05, Pml09, Pml12 (Chirhart et al. ); PLGT15 (Schmidt )]. .. Amplification by multiplex PCR took place in 10 μ L volumes with 20 ng of template DNA, 0.2 mM of each dNTP, 1 U of Taq DNA polymerase (NEB), and 2× Thermopol reaction buffer (20 mM Tris‐HCl, 10 mM (NH4 )2 SO4 , 10 mM KCl, 2 mM MgSO4 , 0.1% Triton X‐100; NEB).

    Article Title: Novel thermostable Y-family polymerases: applications for the PCR amplification of damaged or ancient DNAs
    Article Snippet: PCRs on undamaged DNA For the undamaged plasmid DNA template, 50 µl PCRs were set up containing standard ThermoPol reaction buffer [20 mM Tris–HCl, pH 8.8, 10 mM KCl, 10 mM (NH4 )2 SO4 , 2 mM MgSO4 , 0.1% Triton X-100] (New England Biolabs), 10 ng of circular pJM548 (Ste dpo4 -like in pGEM-T), 200 µM of each ultrapure dNTP and 1 µM of each primer; Ste_FNde5 and Ste_RBam1089 whose sequences are provided above. .. Each of the Dpo4 enzymes was added to a concentration of 200 nM, except the Ssh Dpo4-like enzyme, which was added to a concentration of 400 nM.

    Article Title: A general strategy for expanding polymerase function by droplet microfluidics
    Article Snippet: The primer–template complex was annealed in ThermoPol buffer (1 × : 20 mM Tris-HCl, 10 mM (NH4)2SO4, 10 mM KCl, 2 mM MgSO4, 0.1% Triton X-100, pH 8.8; New England Biolabs) by heating for 5 min at 95 °C and cooling for 5 min at 4 °C. .. The concentration of primer, template and quencher strands were 2 μM, 1 μM and 3 μM, respectively.

    Article Title: Development and application of a rapid and visual loop-mediated isothermal amplification for the detection of Sporisorium scitamineum in sugarcane
    Article Snippet: Thus, the LAMP reaction conditions in combination 7 were optimal for detecting S. scitamineum in sugarcane, which consisted of Mg2+ at a concentration of 6.00 mmol/L, a ratio of inner vs. outer primers of 6:1, and a concentration of Bst DNA polymerase of 8.0 U. .. The optimized LAMP reaction conditions including 4 mmol/L MgSO4 (50 mmol/L), 1.4 mmol/L dNTPs (10 mmol/L), 2.5 μL 10×ThermoPol Reaction Buffer [containing 20 mmol/L Tris-HCl pH 8.8, 10 mmol/L KCl, 2 mmol/L MgSO4 , 10 mmol/L (NH4 )2 SO4 , 0.1% Triton X-100, NEB company], 1.2 μmol/L each of FIP and BIP primers, 0.2 μmol/L each of F3 and B3 primers, 8.0 U Bst DNA polymerase (8 000 U/mL, NEB company), DNA template 1.0 μL, ddH2 O were complemented to a volume of 25 μL.

    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. ..

    Article Title: Strong-LAMP: A LAMP Assay for Strongyloides spp. Detection in Stool and Urine Samples. Towards the Diagnosis of Human Strongyloidiasis Starting from a Rodent Model
    Article Snippet: Paragraph title: Optimization of LAMP assay ... Thus, LAMP reactions mixtures (25 μL) contained 40 pmol of each FIP and BIP primers, 5 pmol of each F3 and B3 primers, 1.4 mM of each dNTP (Bioron), 1x ThermoPol Reaction Buffer -20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4 )2 SO4 , 2 mM MgSO4 , 0.1% Triton X-100; New England Biolabs, UK- (when using Bst Polymerase Large Fragment) or 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- (when using either Bst DNA polymerase 2.0 or Bst DNA polymerase 2.0 WarmStart), betaine (ranging 0.8, 1, 1.2, 1.4 or 1.6 M), supplementary MgSO4 (ranging 2, 4, 6 or 8 mM) and 8 U of the tested Bst polymerase in each case with 2 μL of template DNA.

    Marker:

    Article Title: Rapid and sensitive detection of Pseudocercospora eumusae pathogen causing eumusae leaf spot disease of banana by loop-mediated isothermal amplification (LAMP) method
    Article Snippet: The LAMP primers specific to P. eumusae were designed based on the specific SCAR marker sequence of P. eumusae derived from RAPD analysis. .. To determine the optimal reaction temperature and time, the LAMP reaction mixtures were incubated at 56–65 °C for 15, 30, 45, 60, and 90 min. A total volume of 25 ul for the LAMP reaction contained 0.48 µM FIP and BIP, 0.12 µM concentrations of F3 and B3, 2 mM dNTPs mix, 2.0 U of Bst DNA polymerase large fragment (New England Biolabs, Inc., Beverly, MA), 1× ThermoPol buffer (20 mM Tris–HCl, 10 mM (NH4 )2 SO4 , 10 mM KCl, 2 mM MgSO4 , 0.1% Triton X-100, pH 8.8) (New England Biolabs), 4 mM MgSO4 and 1 µl of template DNA (25 ng).

    Staining:

    Article Title: Evaluating the influence of life‐history characteristics on genetic structure: a comparison of small mammals inhabiting complex agricultural landscapes
    Article Snippet: DNA quality was checked by running 3 μ L of DNA out on a 2% agarose gel stained with ethidium 12 bromide, and then, DNA extracts were diluted to a final concentration of approximately 10 ng/μ L. Chipmunk samples were amplified at 12 (EACH01‐12; Anderson et al. ) microsatellite loci, while white‐footed mice were amplified at 10 loci [PO‐26, PO‐85, Po‐97 (Prince et al. ); Pml01, Pml02, Pml05, Pml09, Pml12 (Chirhart et al. ); PLGT15 (Schmidt )]. .. Amplification by multiplex PCR took place in 10 μ L volumes with 20 ng of template DNA, 0.2 mM of each dNTP, 1 U of Taq DNA polymerase (NEB), and 2× Thermopol reaction buffer (20 mM Tris‐HCl, 10 mM (NH4 )2 SO4 , 10 mM KCl, 2 mM MgSO4 , 0.1% Triton X‐100; NEB).

    Article Title: Novel thermostable Y-family polymerases: applications for the PCR amplification of damaged or ancient DNAs
    Article Snippet: PCRs on undamaged DNA For the undamaged plasmid DNA template, 50 µl PCRs were set up containing standard ThermoPol reaction buffer [20 mM Tris–HCl, pH 8.8, 10 mM KCl, 10 mM (NH4 )2 SO4 , 2 mM MgSO4 , 0.1% Triton X-100] (New England Biolabs), 10 ng of circular pJM548 (Ste dpo4 -like in pGEM-T), 200 µM of each ultrapure dNTP and 1 µM of each primer; Ste_FNde5 and Ste_RBam1089 whose sequences are provided above. .. Reactions were heated to 85°C for 3 min and then treated with 35 cycles of 85°C for 30 s, 56°C for 1 min and 60°C for 4 min. Products were separated on a 0.9% agarose gel stained with ethidium bromide.

    Article Title: Rapid and sensitive detection of Pseudocercospora eumusae pathogen causing eumusae leaf spot disease of banana by loop-mediated isothermal amplification (LAMP) method
    Article Snippet: To determine the optimal reaction temperature and time, the LAMP reaction mixtures were incubated at 56–65 °C for 15, 30, 45, 60, and 90 min. A total volume of 25 ul for the LAMP reaction contained 0.48 µM FIP and BIP, 0.12 µM concentrations of F3 and B3, 2 mM dNTPs mix, 2.0 U of Bst DNA polymerase large fragment (New England Biolabs, Inc., Beverly, MA), 1× ThermoPol buffer (20 mM Tris–HCl, 10 mM (NH4 )2 SO4 , 10 mM KCl, 2 mM MgSO4 , 0.1% Triton X-100, pH 8.8) (New England Biolabs), 4 mM MgSO4 and 1 µl of template DNA (25 ng). .. The visual detection of LAMP products was performed by the addition of 2 µl of 1000× SYBR® Green I nucleic acid gel stain (Invitrogen, Carlsbad, California).

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  • 90
    New England Biolabs lambda phosphatase
    Analysis of PFV Gag phosphorylation status in purified virus particles. PFV virions were produced by transient transfection of the four-component PFV vector system, containing the pcoPG4 variants denoted above each of the blots, into 293T cells. Viral particles were pelleted from cell-free tissue culture supernatants by ultracentrifugation through 20% sucrose and equal amounts of particle lysates separated by SDS-PAGE were blotted to nitrocellulose membranes. The phosphorylation status of the particle-associated Gag variants was determined by the corresponding antibodies specific for different phosphorylated amino acid motives as indicated. Data are representative of n = 4 independent experiments. (A) Schematic illustration of PFV Gag protein organization with highlighted putative T-P motifs and surrounding amino acids recognized when phosphorylated at the threonine residue by either the α-pT-P (orange letters) or α-Gag S-pT-P (blue letters) phosphopeptide-specific antibody. Solid vertical arrow: primary Gag processing site; dashed vertical arrows: secondary Gag processing sites; (B) Detection of the phosphorylated Thr-Pro (pT-P) motives in PFV Gag wt, iSTP and pmSTP virus particles (α-pT-P antiserum) and comparison with the total Gag content in the particle lysates (α-Gag). (C) Comparison of the pT-P phosphorylation status in the wt and T225A PFV particles in the absence (-) or presence (+) of the <t>Lambda</t> Phosphatase (λP) pretreatment of viral proteins. (D) Comparison of the PFV Gag-associated S-T-P motif phosphorylation status (α-Gag S-pT-P; detected with the corresponding PFV Gag-specific antibody) in virus preparations containing either the wt or the T225A Gag variant in the absence (-) or presence (+) of the λP pretreatment.
    Lambda Phosphatase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 90/100, based on 32 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs triton x 100
    Analysis of PFV Gag phosphorylation status in purified virus particles. PFV virions were produced by transient transfection of the four-component PFV vector system, containing the pcoPG4 variants denoted above each of the blots, into 293T cells. Viral particles were pelleted from cell-free tissue culture supernatants by ultracentrifugation through 20% sucrose and equal amounts of particle lysates separated by SDS-PAGE were blotted to nitrocellulose membranes. The phosphorylation status of the particle-associated Gag variants was determined by the corresponding antibodies specific for different phosphorylated amino acid motives as indicated. Data are representative of n = 4 independent experiments. (A) Schematic illustration of PFV Gag protein organization with highlighted putative T-P motifs and surrounding amino acids recognized when phosphorylated at the threonine residue by either the α-pT-P (orange letters) or α-Gag S-pT-P (blue letters) phosphopeptide-specific antibody. Solid vertical arrow: primary Gag processing site; dashed vertical arrows: secondary Gag processing sites; (B) Detection of the phosphorylated Thr-Pro (pT-P) motives in PFV Gag wt, iSTP and pmSTP virus particles (α-pT-P antiserum) and comparison with the total Gag content in the particle lysates (α-Gag). (C) Comparison of the pT-P phosphorylation status in the wt and T225A PFV particles in the absence (-) or presence (+) of the <t>Lambda</t> Phosphatase (λP) pretreatment of viral proteins. (D) Comparison of the PFV Gag-associated S-T-P motif phosphorylation status (α-Gag S-pT-P; detected with the corresponding PFV Gag-specific antibody) in virus preparations containing either the wt or the T225A Gag variant in the absence (-) or presence (+) of the λP pretreatment.
    Triton X 100, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 90/100, based on 49 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs vanadyl ribonucleoside complex
    Analysis of PFV Gag phosphorylation status in purified virus particles. PFV virions were produced by transient transfection of the four-component PFV vector system, containing the pcoPG4 variants denoted above each of the blots, into 293T cells. Viral particles were pelleted from cell-free tissue culture supernatants by ultracentrifugation through 20% sucrose and equal amounts of particle lysates separated by SDS-PAGE were blotted to nitrocellulose membranes. The phosphorylation status of the particle-associated Gag variants was determined by the corresponding antibodies specific for different phosphorylated amino acid motives as indicated. Data are representative of n = 4 independent experiments. (A) Schematic illustration of PFV Gag protein organization with highlighted putative T-P motifs and surrounding amino acids recognized when phosphorylated at the threonine residue by either the α-pT-P (orange letters) or α-Gag S-pT-P (blue letters) phosphopeptide-specific antibody. Solid vertical arrow: primary Gag processing site; dashed vertical arrows: secondary Gag processing sites; (B) Detection of the phosphorylated Thr-Pro (pT-P) motives in PFV Gag wt, iSTP and pmSTP virus particles (α-pT-P antiserum) and comparison with the total Gag content in the particle lysates (α-Gag). (C) Comparison of the pT-P phosphorylation status in the wt and T225A PFV particles in the absence (-) or presence (+) of the <t>Lambda</t> Phosphatase (λP) pretreatment of viral proteins. (D) Comparison of the PFV Gag-associated S-T-P motif phosphorylation status (α-Gag S-pT-P; detected with the corresponding PFV Gag-specific antibody) in virus preparations containing either the wt or the T225A Gag variant in the absence (-) or presence (+) of the λP pretreatment.
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    Analysis of PFV Gag phosphorylation status in purified virus particles. PFV virions were produced by transient transfection of the four-component PFV vector system, containing the pcoPG4 variants denoted above each of the blots, into 293T cells. Viral particles were pelleted from cell-free tissue culture supernatants by ultracentrifugation through 20% sucrose and equal amounts of particle lysates separated by SDS-PAGE were blotted to nitrocellulose membranes. The phosphorylation status of the particle-associated Gag variants was determined by the corresponding antibodies specific for different phosphorylated amino acid motives as indicated. Data are representative of n = 4 independent experiments. (A) Schematic illustration of PFV Gag protein organization with highlighted putative T-P motifs and surrounding amino acids recognized when phosphorylated at the threonine residue by either the α-pT-P (orange letters) or α-Gag S-pT-P (blue letters) phosphopeptide-specific antibody. Solid vertical arrow: primary Gag processing site; dashed vertical arrows: secondary Gag processing sites; (B) Detection of the phosphorylated Thr-Pro (pT-P) motives in PFV Gag wt, iSTP and pmSTP virus particles (α-pT-P antiserum) and comparison with the total Gag content in the particle lysates (α-Gag). (C) Comparison of the pT-P phosphorylation status in the wt and T225A PFV particles in the absence (-) or presence (+) of the Lambda Phosphatase (λP) pretreatment of viral proteins. (D) Comparison of the PFV Gag-associated S-T-P motif phosphorylation status (α-Gag S-pT-P; detected with the corresponding PFV Gag-specific antibody) in virus preparations containing either the wt or the T225A Gag variant in the absence (-) or presence (+) of the λP pretreatment.

    Journal: PLoS Pathogens

    Article Title: Interactions of Prototype Foamy Virus Capsids with Host Cell Polo-Like Kinases Are Important for Efficient Viral DNA Integration

    doi: 10.1371/journal.ppat.1005860

    Figure Lengend Snippet: Analysis of PFV Gag phosphorylation status in purified virus particles. PFV virions were produced by transient transfection of the four-component PFV vector system, containing the pcoPG4 variants denoted above each of the blots, into 293T cells. Viral particles were pelleted from cell-free tissue culture supernatants by ultracentrifugation through 20% sucrose and equal amounts of particle lysates separated by SDS-PAGE were blotted to nitrocellulose membranes. The phosphorylation status of the particle-associated Gag variants was determined by the corresponding antibodies specific for different phosphorylated amino acid motives as indicated. Data are representative of n = 4 independent experiments. (A) Schematic illustration of PFV Gag protein organization with highlighted putative T-P motifs and surrounding amino acids recognized when phosphorylated at the threonine residue by either the α-pT-P (orange letters) or α-Gag S-pT-P (blue letters) phosphopeptide-specific antibody. Solid vertical arrow: primary Gag processing site; dashed vertical arrows: secondary Gag processing sites; (B) Detection of the phosphorylated Thr-Pro (pT-P) motives in PFV Gag wt, iSTP and pmSTP virus particles (α-pT-P antiserum) and comparison with the total Gag content in the particle lysates (α-Gag). (C) Comparison of the pT-P phosphorylation status in the wt and T225A PFV particles in the absence (-) or presence (+) of the Lambda Phosphatase (λP) pretreatment of viral proteins. (D) Comparison of the PFV Gag-associated S-T-P motif phosphorylation status (α-Gag S-pT-P; detected with the corresponding PFV Gag-specific antibody) in virus preparations containing either the wt or the T225A Gag variant in the absence (-) or presence (+) of the λP pretreatment.

    Article Snippet: Subsequently, half of the sample was digested for 1 h at 30°C with Lambda Phosphatase (NEB, P0753; 7.15 U/μl Lambda PP, 1% Triton X-100, 1 mM MnCl2 , 1x Lambda Phosphatase buffer) whereas the other half was mock digested prior to addition of protein sample buffer.

    Techniques: Purification, Produced, Transfection, Plasmid Preparation, SDS Page, Variant Assay