afliii  (New England Biolabs)


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    Name:
    AflIII
    Description:
    AflIII 1 250 units
    Catalog Number:
    r0541l
    Price:
    282
    Size:
    1 250 units
    Category:
    Restriction Enzymes
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    Structured Review

    New England Biolabs afliii
    AflIII
    AflIII 1 250 units
    https://www.bioz.com/result/afliii/product/New England Biolabs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    afliii - by Bioz Stars, 2020-02
    95/100 stars

    Images

    1) Product Images from "A tale of two haplotype groups: Evaluating the New World Junonia ring species hypothesis using the distribution of divergent COI haplotypes"

    Article Title: A tale of two haplotype groups: Evaluating the New World Junonia ring species hypothesis using the distribution of divergent COI haplotypes

    Journal: Systematic entomology

    doi: 10.1111/syen.12120

    Map of PCR primer binding sites and restriction enzyme cut sizes for the barcode fragment of COI. BamHI restriction sites are only found in haplotype group A alleles while AflIII and BseYI restriction sites are only found in haplotype group B alleles.
    Figure Legend Snippet: Map of PCR primer binding sites and restriction enzyme cut sizes for the barcode fragment of COI. BamHI restriction sites are only found in haplotype group A alleles while AflIII and BseYI restriction sites are only found in haplotype group B alleles.

    Techniques Used: Polymerase Chain Reaction, Binding Assay

    2) Product Images from "Development and validation of a multiplex-PCR assay for X-linked intellectual disability"

    Article Title: Development and validation of a multiplex-PCR assay for X-linked intellectual disability

    Journal: BMC Medical Genetics

    doi: 10.1186/1471-2350-14-80

    Examples of Southern blot results. Genotyping using FMR1 probe: After EcoRI /EagI double digestion, normal females exhibit two fragments, a ~2.8 kb (active X) and a ~5.2 kb (inactive X), while in normal males only a ~2.8 kb fragment is seen: (a) FXS male mosaic for a 51 CGG allele and a full mutation; (b) Premutated FMR1 male (110 CGGs); (c) Klinefelter with two FMR1 normal sized-alleles, showing the active and the inactive X-chromosome; (d) Normal FMR1 male (19 CGGs); (e) Premutated FMR1 male (58 CGGs). Genotyping using AFF2 probe: Following AflIII /NotI double digestion, normal females present two fragments, a ~2.2 kb (active X) and a ~4.8 kb (inactive X), whereas normal males exhibit only a ~2.2 kb fragment: (f) Intermediate AFF2 male (47 CCGs); (g) Premutated AFF2 male (68 CCGs); (h) Homoallelic female [(CCG) 14 /(CCG) 14 ].
    Figure Legend Snippet: Examples of Southern blot results. Genotyping using FMR1 probe: After EcoRI /EagI double digestion, normal females exhibit two fragments, a ~2.8 kb (active X) and a ~5.2 kb (inactive X), while in normal males only a ~2.8 kb fragment is seen: (a) FXS male mosaic for a 51 CGG allele and a full mutation; (b) Premutated FMR1 male (110 CGGs); (c) Klinefelter with two FMR1 normal sized-alleles, showing the active and the inactive X-chromosome; (d) Normal FMR1 male (19 CGGs); (e) Premutated FMR1 male (58 CGGs). Genotyping using AFF2 probe: Following AflIII /NotI double digestion, normal females present two fragments, a ~2.2 kb (active X) and a ~4.8 kb (inactive X), whereas normal males exhibit only a ~2.2 kb fragment: (f) Intermediate AFF2 male (47 CCGs); (g) Premutated AFF2 male (68 CCGs); (h) Homoallelic female [(CCG) 14 /(CCG) 14 ].

    Techniques Used: Southern Blot, Mutagenesis

    3) Product Images from "Low-cost production of proinsulin in tobacco and lettuce chloroplasts for injectable or oral delivery of functional insulin and C-peptide"

    Article Title: Low-cost production of proinsulin in tobacco and lettuce chloroplasts for injectable or oral delivery of functional insulin and C-peptide

    Journal: Plant biotechnology journal

    doi: 10.1111/j.1467-7652.2010.00582.x

    Southern analysis of transgenic tobacco containing cholera toxin B subunit (CTB)-Proinsulin with three furin cleavage sites. (a) Map of untransformed tobacco plastome. Digestion with AflIII yields a 4.2-kb fragment. Broken line represents probe annealing site. (b) Map of transformed tobacco plastome. Digestion with AflIII yields a 6.4-kb fragment. The 5′UTR represents the light-regulated psbA promoter. (c) Southern analysis from T 1 generation. A and B represent independent transplastomic lines. WT, untransformed plant.
    Figure Legend Snippet: Southern analysis of transgenic tobacco containing cholera toxin B subunit (CTB)-Proinsulin with three furin cleavage sites. (a) Map of untransformed tobacco plastome. Digestion with AflIII yields a 4.2-kb fragment. Broken line represents probe annealing site. (b) Map of transformed tobacco plastome. Digestion with AflIII yields a 6.4-kb fragment. The 5′UTR represents the light-regulated psbA promoter. (c) Southern analysis from T 1 generation. A and B represent independent transplastomic lines. WT, untransformed plant.

    Techniques Used: Transgenic Assay, CtB Assay, Transformation Assay

    4) Product Images from "The mechanism of DNA replication termination in vertebrates"

    Article Title: The mechanism of DNA replication termination in vertebrates

    Journal: Nature

    doi: 10.1038/nature14887

    DNA synthesis does not stall during termination A. Cartoon depicting the assay for lacO array synthesis. B. LacR Block-IPTG release was performed on p[ lacO x12]. To measure synthesis within the array, termination intermediates were cut with AflIII and PvuII to liberate the array fragment from the vector. Cleaved products were separated by native gel electrophoresis. Different exposures of array and vector fragments are shown (see methods). C. Array synthesis, vector synthesis, and dissolution were quantified. Means±s.d. are plotted (n=3).
    Figure Legend Snippet: DNA synthesis does not stall during termination A. Cartoon depicting the assay for lacO array synthesis. B. LacR Block-IPTG release was performed on p[ lacO x12]. To measure synthesis within the array, termination intermediates were cut with AflIII and PvuII to liberate the array fragment from the vector. Cleaved products were separated by native gel electrophoresis. Different exposures of array and vector fragments are shown (see methods). C. Array synthesis, vector synthesis, and dissolution were quantified. Means±s.d. are plotted (n=3).

    Techniques Used: DNA Synthesis, Blocking Assay, Plasmid Preparation, Nucleic Acid Electrophoresis

    Sequence-specific termination can be induced at a LacR array (A) To investigate whether a LacR array blocks replication forks, a plasmid containing a tandem array of 16 lac operator ( lacO ) sequences, p[ lacO x16], was incubated with buffer or LacR and then replicated in egg extract containing [α- 32 P]dATP. Radiolabelled replication intermediates were cleaved with XmnI (far left cartoon) and separated according to size and shape by 2-dimensional gel electrophoresis (see schematic of 2-D gel). As replication neared completion at 4.5 minutes, mainly linear molecules were produced in the presence of buffer (orange arrowhead). In contrast, in the presence of LacR, a discrete spot appeared on the Double Y arc (blue arrowhead), demonstrating that converging replication forks accumulate at a specific locus on p[ lacO x16]. These data indicate that 16 copies of LacR block replication forks. (B–F) To test whether the double-Y structures observed in (A) arose from replication forks stalling at the outer edges of the lacO array, we tested whether LacR specifically inhibited replication of lacO sequences. To this end, p[ lacOx16 ] (C) and the parental plasmid lacking lacO repeats, p[ empty ] (B), were incubated in the presence of buffer or LacR and replicated using Xenopus egg extracts containing [α- 32 P]dATP. Radiolabelled replication intermediates were cleaved with AflIII and PvuII to release the 2354 bp plasmid backbone (B and C) and a 294 bp control fragment from p[ empty ] (B) or a 794 bp lacO fragment from p[ lacO x16] (C). The plasmid backbone and the respective inserts were separated on a native gel and detected by autoradiography (D). A longer exposure of the small fragments is shown, since they are less intense than the large fragments. The results in panel (D) were quantified in (E) and (F). Importantly, LacR specifically inhibited replication of the lacO -containing fragment in p[ lacO x16] (F, blue circles) but not the control fragment in p[ empty ] (E, green circles). We conclude that LacR prevents replication of the lacO array and that the double-Y’s in (A) represent forks converged on the outer edges of the array. Importantly, synthesis within the 2354 bp backbone fragment (F, orange circles) of p[ lacO x16] was not inhibited in the presence of LacR, indicating that no global structural changes occur that inhibit replication.
    Figure Legend Snippet: Sequence-specific termination can be induced at a LacR array (A) To investigate whether a LacR array blocks replication forks, a plasmid containing a tandem array of 16 lac operator ( lacO ) sequences, p[ lacO x16], was incubated with buffer or LacR and then replicated in egg extract containing [α- 32 P]dATP. Radiolabelled replication intermediates were cleaved with XmnI (far left cartoon) and separated according to size and shape by 2-dimensional gel electrophoresis (see schematic of 2-D gel). As replication neared completion at 4.5 minutes, mainly linear molecules were produced in the presence of buffer (orange arrowhead). In contrast, in the presence of LacR, a discrete spot appeared on the Double Y arc (blue arrowhead), demonstrating that converging replication forks accumulate at a specific locus on p[ lacO x16]. These data indicate that 16 copies of LacR block replication forks. (B–F) To test whether the double-Y structures observed in (A) arose from replication forks stalling at the outer edges of the lacO array, we tested whether LacR specifically inhibited replication of lacO sequences. To this end, p[ lacOx16 ] (C) and the parental plasmid lacking lacO repeats, p[ empty ] (B), were incubated in the presence of buffer or LacR and replicated using Xenopus egg extracts containing [α- 32 P]dATP. Radiolabelled replication intermediates were cleaved with AflIII and PvuII to release the 2354 bp plasmid backbone (B and C) and a 294 bp control fragment from p[ empty ] (B) or a 794 bp lacO fragment from p[ lacO x16] (C). The plasmid backbone and the respective inserts were separated on a native gel and detected by autoradiography (D). A longer exposure of the small fragments is shown, since they are less intense than the large fragments. The results in panel (D) were quantified in (E) and (F). Importantly, LacR specifically inhibited replication of the lacO -containing fragment in p[ lacO x16] (F, blue circles) but not the control fragment in p[ empty ] (E, green circles). We conclude that LacR prevents replication of the lacO array and that the double-Y’s in (A) represent forks converged on the outer edges of the array. Importantly, synthesis within the 2354 bp backbone fragment (F, orange circles) of p[ lacO x16] was not inhibited in the presence of LacR, indicating that no global structural changes occur that inhibit replication.

    Techniques Used: Sequencing, Plasmid Preparation, Incubation, Nucleic Acid Electrophoresis, Produced, Blocking Assay, Autoradiography

    Related Articles

    Clone Assay:

    Article Title: IMAGEtags: Quantifying mRNA transcription in real time with multiaptamer reporters
    Article Snippet: Paragraph title: Cloning initial repetitive aptamer sequences from synthetic oligonucleotides ... Digest pZErO-2 vector with Afl III (New England Biolabs-NEB, Ipswich, MA, Cat# R0541S) and Apo I (NEB, R0566S) restriction enzymes and isolate the 1854 base pair fragment using gel electrophoresis followed by extraction and purification by a method such as the QIAquick gel extraction kit (QIAGEN, Valencia, CA, Cat# 28704).

    Amplification:

    Article Title: Artesunate-Amodiaquine and Artemether-Lumefantrine Therapies and Selection of Pfcrt and Pfmdr1 Alleles in Nanoro, Burkina Faso
    Article Snippet: Briefly, an initial amplification of the outer region of each gene was followed by nested PCR using specific primers. .. For Pfmdr1 the following restriction enzymes were used: Afl III (NEB), Dra I (NEB), Dde I (NEB), Ase I (NEB), and Eco R V (NEB) respectively for Pfmdr1-N86Y , Pfmdr1 -Y184F, Pfmdr1 -S1034C, Pfmdr1 - N1042D and, Pfmdr1 -D1246Y.

    Article Title: Asymptomatic malaria infections and Pfmdr1 mutations in an endemic area of Nigeria
    Article Snippet: PCR amplification was done on C1000 Touch™ Thermal Cycler (Bio-Rad Laboratories Inc., USA) using PCR super-mix (New England Biolabs, USA). .. Nested PCR products were restricted by enzymatic digest with specific restriction enzymes: Afl III (New England Biolabs, USA) was used to detect mutation causing base change from asparagine (N) to tyrosine (Y) at codon 86 while EcoRV (Beijing TransGen Biotech, China) was used to detect mutation causing base change from aspartic acid (D) to tyrosine (N) at codon 1246.

    Article Title: Erratum to: ‘The Memory Aid study: protocol for a randomized controlled clinical trial evaluating the effect of computer-based working memory training in elderly patients with mild cognitive impairment (MCI)’
    Article Snippet: .. Then 15 μl of the amplification PCR products will be digested directly by 2.5 U of restriction enzymes MslI (R0571S, New England Biolabs, Beverly, MA) for 2 hours at 37 °C, and by Hae II (R0107S, New England Biolabs, Beverly, MA, USA) and Afl III (R0541S, New England Biolabs, Beverly, MA, USA) overnight at 37 °C. .. The digested PCR products will then be analyzed on 4 % agarose gel and visualized using GelGreen™ Nucleic Acid Gel Stain (89139–144, Biotium, Hayward, CA).

    Article Title: Extraction of amplifiable DNA from embalmed human cadaver tissue
    Article Snippet: .. Amplified DNA was digested with HaeII and AflIII (NEB cat#R0107S and R0541S) for 2 h at 37 °C and resolved on a 4% agarose gel. ..

    Article Title: Sequence analysis of pfcrt and pfmdr1 genes and its association with chloroquine resistance in Southeast Indian Plasmodium falciparum isolates
    Article Snippet: .. 5.4 Restriction digestion with Apo I and Afl III The final PCR amplified product of pfcrt and pfmdr1 gene was enzymatically digested with Apo I and Afl III (New England Biolabs, UK) respectively. .. Digestion of the pfcrt PCR product into two fragments (128 & 136 bp) with Apo I enzyme confers that the isolate was sensitive to CQ with CVMNK haplotype, whereas, the K76 T mutation was resistant to digestion .

    Article Title: Correlation of Molecular Markers, Pfmdr1-N86Y and Pfcrt-K76T, with In Vitro Chloroquine Resistant Plasmodium falciparum, Isolated in the Malaria Endemic States of Assam and Arunachal Pradesh, Northeast India
    Article Snippet: .. Restriction Digestion with ApoI and Afl III The finally amplified product was subjected to restriction digestion with Afl III (mutational allele) and Apo I (wild type allele) (New England Biolabs, UK) by incubating at 37°C for one hour with the one unit of each enzyme. .. The digests were resolved on 3% agarose gel, stained with ethidium bromide, and results were recorded on the gel documentation system (UVITEC, UK).

    Binding Assay:

    Article Title: Signaling from DNA mispairs to mismatch-repair excision sites despite intervening blockades
    Article Snippet: Paragraph title: Bandshift assay for hMutSα binding ... As described previously , 0.5-kb fragments were cleaved from non-nicked substrates E or its homoduplex homolog with endonuclease Bgl II and Afl III, electrophoretically purified, and endlabeled by filling in the recessed 3′ ends using 32 P-α-dCTP and three other non-radioactive dNTPs in the presence of DNA polI Klenow fragment (exo− ) (New England Biolab).

    Article Title: Designing and overproducing a tandem epitope of gp350/220 that shows a potential to become an EBV vaccine
    Article Snippet: The sequence was inserted in the downstream of Maltose binding Protein (MBP), and just between Factor Xa in upstream and rrnB T1 terminator sequence. .. The insertion results were checked for validity based on the excision of the restriction enzyme (AflIII; NEB Cat No. R0541S).

    Synthesized:

    Article Title: Designing and overproducing a tandem epitope of gp350/220 that shows a potential to become an EBV vaccine
    Article Snippet: The sequence encoding the tandem epitope was synthesized by Genescript and inserted into pMAL-p5x plasmid. .. The insertion results were checked for validity based on the excision of the restriction enzyme (AflIII; NEB Cat No. R0541S).

    Construct:

    Article Title: Designing and overproducing a tandem epitope of gp350/220 that shows a potential to become an EBV vaccine
    Article Snippet: The insertion results were checked for validity based on the excision of the restriction enzyme (AflIII; NEB Cat No. R0541S). .. Following, the E. c oli strain BL21 was transformed using a newly constructed plasmid.

    Electrophoresis:

    Article Title: Signaling from DNA mispairs to mismatch-repair excision sites despite intervening blockades
    Article Snippet: As described previously , 0.5-kb fragments were cleaved from non-nicked substrates E or its homoduplex homolog with endonuclease Bgl II and Afl III, electrophoretically purified, and endlabeled by filling in the recessed 3′ ends using 32 P-α-dCTP and three other non-radioactive dNTPs in the presence of DNA polI Klenow fragment (exo− ) (New England Biolab). .. After addition of 1/5 volume of 50% sucrose, products were separated by electrophoresis through 5% nondenaturing polyacrylamide gels (acrylamide:bis-acrylamide ratio at 37.5:1) as described ( ).

    Incubation:

    Article Title: Signaling from DNA mispairs to mismatch-repair excision sites despite intervening blockades
    Article Snippet: As described previously , 0.5-kb fragments were cleaved from non-nicked substrates E or its homoduplex homolog with endonuclease Bgl II and Afl III, electrophoretically purified, and endlabeled by filling in the recessed 3′ ends using 32 P-α-dCTP and three other non-radioactive dNTPs in the presence of DNA polI Klenow fragment (exo− ) (New England Biolab). .. After standard incubation with streptavidin in Binding Buffer, where indicated, these fragments, plus 125 ng of 1-kb ladder DNA (Invitrogen, catalog number 15615-016), were incubated with purified hMutSα (10 min), then with ATP (5 min), where indicated.

    Article Title: Detection of DNA point mutations and mRNA expression levels by rolling circle amplification in individual cells
    Article Snippet: Either Afl III or Bbs I (0.1 unit/μl, New England Biolabs) was applied for 12 h at 37°C. .. Cells were treated with exonuclease III (1.3 units/μl, Life Technologies, Grand Island, NY) in 1× exonuclease III buffer (50 mM Tris, pH 8.0/5 mM MgCl2 /1 mM DTT), then incubated 1 h at 37°C and rinsed with sterile water.

    Expressing:

    Article Title: Detection of DNA point mutations and mRNA expression levels by rolling circle amplification in individual cells
    Article Snippet: HLB cells were expected to have two normal copies of the Tp53 gene and to be normal with regard to Tp53 expression. .. Either Afl III or Bbs I (0.1 unit/μl, New England Biolabs) was applied for 12 h at 37°C.

    Transformation Assay:

    Article Title: Designing and overproducing a tandem epitope of gp350/220 that shows a potential to become an EBV vaccine
    Article Snippet: Paragraph title: 2.5. Transformation and recombinant protein overexpression ... The insertion results were checked for validity based on the excision of the restriction enzyme (AflIII; NEB Cat No. R0541S).

    Over Expression:

    Article Title: Designing and overproducing a tandem epitope of gp350/220 that shows a potential to become an EBV vaccine
    Article Snippet: Paragraph title: 2.5. Transformation and recombinant protein overexpression ... The insertion results were checked for validity based on the excision of the restriction enzyme (AflIII; NEB Cat No. R0541S).

    Ligation:

    Article Title: A Rapid and Economic In-House DNA Purification Method Using Glass Syringe Filters
    Article Snippet: .. Enzyme Digestion and Ligation Bgl II, Afl III, Cla I, and ApaL I (NEB) were used to digest 3 ug of pLL3.7 and pLL-LS at 37°C for one hour in 60 ul using the manufacture's recommended buffer. ..

    Polymerase Chain Reaction:

    Article Title: Artesunate-Amodiaquine and Artemether-Lumefantrine Therapies and Selection of Pfcrt and Pfmdr1 Alleles in Nanoro, Burkina Faso
    Article Snippet: For Pfcrt the restriction enzyme Apo I was used for the digestion of PCR products. .. For Pfmdr1 the following restriction enzymes were used: Afl III (NEB), Dra I (NEB), Dde I (NEB), Ase I (NEB), and Eco R V (NEB) respectively for Pfmdr1-N86Y , Pfmdr1 -Y184F, Pfmdr1 -S1034C, Pfmdr1 - N1042D and, Pfmdr1 -D1246Y.

    Article Title: Erratum to: ‘The Memory Aid study: protocol for a randomized controlled clinical trial evaluating the effect of computer-based working memory training in elderly patients with mild cognitive impairment (MCI)’
    Article Snippet: .. Then 15 μl of the amplification PCR products will be digested directly by 2.5 U of restriction enzymes MslI (R0571S, New England Biolabs, Beverly, MA) for 2 hours at 37 °C, and by Hae II (R0107S, New England Biolabs, Beverly, MA, USA) and Afl III (R0541S, New England Biolabs, Beverly, MA, USA) overnight at 37 °C. .. The digested PCR products will then be analyzed on 4 % agarose gel and visualized using GelGreen™ Nucleic Acid Gel Stain (89139–144, Biotium, Hayward, CA).

    Article Title: Light-dependent regulation of sleep/wake states by prokineticin 2 in zebrafish
    Article Snippet: .. PCR products were digested with AflIII (R0541, New England Biolabs Inc.). .. Mutant (527 bp) and WT (218 bp + 316 bp) bands were distinguished by running the digested PCR reaction on a 2% agarose gel.

    Article Title: Extraction of amplifiable DNA from embalmed human cadaver tissue
    Article Snippet: Paragraph title: APOE PCR amplification and digest ... Amplified DNA was digested with HaeII and AflIII (NEB cat#R0107S and R0541S) for 2 h at 37 °C and resolved on a 4% agarose gel.

    Article Title: Sequence analysis of pfcrt and pfmdr1 genes and its association with chloroquine resistance in Southeast Indian Plasmodium falciparum isolates
    Article Snippet: .. 5.4 Restriction digestion with Apo I and Afl III The final PCR amplified product of pfcrt and pfmdr1 gene was enzymatically digested with Apo I and Afl III (New England Biolabs, UK) respectively. .. Digestion of the pfcrt PCR product into two fragments (128 & 136 bp) with Apo I enzyme confers that the isolate was sensitive to CQ with CVMNK haplotype, whereas, the K76 T mutation was resistant to digestion .

    Article Title: Sequence Diversity of pfmdr1 and Sequence Conserve of pldh in Plasmodium falciparum from Indonesia: Its implications on Designing a Novel Antimalarial Drug with Less Prone to Resistance
    Article Snippet: .. Molecular analyses of pfmdr1 RFLP analysis of pfmdr1 codon 86 and 1034 were conducted by digestion of the PCR product with Afl III and Dde I (New England Biolabs, Beverly, MA), respectivelly, at 37o C for 1 h. For each locus , RFLP products were electrophoresed on 1% agarose gels and visualized by UV transillumination. .. Amplification of pldh Oligonucleotide primers pLDH S Kpn and pLDH AS Eco corresponding to pfldh open reading frame (ORF) were constructed based on pfldh sequence (K1 strain) ( ).

    Recombinant:

    Article Title: Designing and overproducing a tandem epitope of gp350/220 that shows a potential to become an EBV vaccine
    Article Snippet: Paragraph title: 2.5. Transformation and recombinant protein overexpression ... The insertion results were checked for validity based on the excision of the restriction enzyme (AflIII; NEB Cat No. R0541S).

    Nucleic Acid Electrophoresis:

    Article Title: IMAGEtags: Quantifying mRNA transcription in real time with multiaptamer reporters
    Article Snippet: .. Digest pZErO-2 vector with Afl III (New England Biolabs-NEB, Ipswich, MA, Cat# R0541S) and Apo I (NEB, R0566S) restriction enzymes and isolate the 1854 base pair fragment using gel electrophoresis followed by extraction and purification by a method such as the QIAquick gel extraction kit (QIAGEN, Valencia, CA, Cat# 28704). .. Use two pairs of complementary oligonucleotides to create inserts containing between two and seven repeats (150–250bp) of the sequences flanked by restriction sites for Bsa I (NEB, R0535S) and Bsm AI (NEB, R0529S) for subsequent multiplication ( ).

    Mutagenesis:

    Article Title: Artesunate-Amodiaquine and Artemether-Lumefantrine Therapies and Selection of Pfcrt and Pfmdr1 Alleles in Nanoro, Burkina Faso
    Article Snippet: For Pfmdr1 the following restriction enzymes were used: Afl III (NEB), Dra I (NEB), Dde I (NEB), Ase I (NEB), and Eco R V (NEB) respectively for Pfmdr1-N86Y , Pfmdr1 -Y184F, Pfmdr1 -S1034C, Pfmdr1 - N1042D and, Pfmdr1 -D1246Y. .. After digestion, DNA bands were visualized in ethidium bromide-stained 2.5% agarose gels for 2 hours at 80 V. 3D7 were used as wild type control for both Pfcrt (codon 76) and Pfmdr1 and the following mutant controls were used: Dd2 (Pfmdr1 -N86Y), 7G8 (Pfmdr1 Y184F/S1034C/N1042D/D1246Y, Pfcrt -K76T).

    Article Title: Asymptomatic malaria infections and Pfmdr1 mutations in an endemic area of Nigeria
    Article Snippet: .. Nested PCR products were restricted by enzymatic digest with specific restriction enzymes: Afl III (New England Biolabs, USA) was used to detect mutation causing base change from asparagine (N) to tyrosine (Y) at codon 86 while EcoRV (Beijing TransGen Biotech, China) was used to detect mutation causing base change from aspartic acid (D) to tyrosine (N) at codon 1246. ..

    Article Title: Light-dependent regulation of sleep/wake states by prokineticin 2 in zebrafish
    Article Snippet: Paragraph title: prokr1 (ENSDARG00000074182, also known as prokr1a ) ct843 mutant ... PCR products were digested with AflIII (R0541, New England Biolabs Inc.).

    Article Title: Sequence analysis of pfcrt and pfmdr1 genes and its association with chloroquine resistance in Southeast Indian Plasmodium falciparum isolates
    Article Snippet: 5.4 Restriction digestion with Apo I and Afl III The final PCR amplified product of pfcrt and pfmdr1 gene was enzymatically digested with Apo I and Afl III (New England Biolabs, UK) respectively. .. Digestion of the pfcrt PCR product into two fragments (128 & 136 bp) with Apo I enzyme confers that the isolate was sensitive to CQ with CVMNK haplotype, whereas, the K76 T mutation was resistant to digestion .

    Microscopy:

    Article Title: Detection of DNA point mutations and mRNA expression levels by rolling circle amplification in individual cells
    Article Snippet: Cells were prepared for DNA detection by first incubating in a hypotonic solution (0.075 M KCl) for 30 min at 37°C followed by three fixations in methanol/acetic acid (3:1 vol/vol) and dropped on clean glass microscope slides. .. Either Afl III or Bbs I (0.1 unit/μl, New England Biolabs) was applied for 12 h at 37°C.

    Purification:

    Article Title: Signaling from DNA mispairs to mismatch-repair excision sites despite intervening blockades
    Article Snippet: .. As described previously , 0.5-kb fragments were cleaved from non-nicked substrates E or its homoduplex homolog with endonuclease Bgl II and Afl III, electrophoretically purified, and endlabeled by filling in the recessed 3′ ends using 32 P-α-dCTP and three other non-radioactive dNTPs in the presence of DNA polI Klenow fragment (exo− ) (New England Biolab). .. After standard incubation with streptavidin in Binding Buffer, where indicated, these fragments, plus 125 ng of 1-kb ladder DNA (Invitrogen, catalog number 15615-016), were incubated with purified hMutSα (10 min), then with ATP (5 min), where indicated.

    Article Title: IMAGEtags: Quantifying mRNA transcription in real time with multiaptamer reporters
    Article Snippet: .. Digest pZErO-2 vector with Afl III (New England Biolabs-NEB, Ipswich, MA, Cat# R0541S) and Apo I (NEB, R0566S) restriction enzymes and isolate the 1854 base pair fragment using gel electrophoresis followed by extraction and purification by a method such as the QIAquick gel extraction kit (QIAGEN, Valencia, CA, Cat# 28704). .. Use two pairs of complementary oligonucleotides to create inserts containing between two and seven repeats (150–250bp) of the sequences flanked by restriction sites for Bsa I (NEB, R0535S) and Bsm AI (NEB, R0529S) for subsequent multiplication ( ).

    Sequencing:

    Article Title: Asymptomatic malaria infections and Pfmdr1 mutations in an endemic area of Nigeria
    Article Snippet: Nested PCR products were restricted by enzymatic digest with specific restriction enzymes: Afl III (New England Biolabs, USA) was used to detect mutation causing base change from asparagine (N) to tyrosine (Y) at codon 86 while EcoRV (Beijing TransGen Biotech, China) was used to detect mutation causing base change from aspartic acid (D) to tyrosine (N) at codon 1246. .. To confirm mutation, 15 µl of amplicons were sent for sequencing (Inqaba Biotec West Africa Ltd, South Africa).

    Article Title: Detection of DNA point mutations and mRNA expression levels by rolling circle amplification in individual cells
    Article Snippet: Restriction enzymes were used to cut ≈20 base pairs either 3′ or 5′ of the sequence of interest, Tp53 in this case. .. Either Afl III or Bbs I (0.1 unit/μl, New England Biolabs) was applied for 12 h at 37°C.

    Article Title: Designing and overproducing a tandem epitope of gp350/220 that shows a potential to become an EBV vaccine
    Article Snippet: The sequence was inserted in the downstream of Maltose binding Protein (MBP), and just between Factor Xa in upstream and rrnB T1 terminator sequence. .. The insertion results were checked for validity based on the excision of the restriction enzyme (AflIII; NEB Cat No. R0541S).

    Gel Extraction:

    Article Title: IMAGEtags: Quantifying mRNA transcription in real time with multiaptamer reporters
    Article Snippet: .. Digest pZErO-2 vector with Afl III (New England Biolabs-NEB, Ipswich, MA, Cat# R0541S) and Apo I (NEB, R0566S) restriction enzymes and isolate the 1854 base pair fragment using gel electrophoresis followed by extraction and purification by a method such as the QIAquick gel extraction kit (QIAGEN, Valencia, CA, Cat# 28704). .. Use two pairs of complementary oligonucleotides to create inserts containing between two and seven repeats (150–250bp) of the sequences flanked by restriction sites for Bsa I (NEB, R0535S) and Bsm AI (NEB, R0529S) for subsequent multiplication ( ).

    Nested PCR:

    Article Title: Artesunate-Amodiaquine and Artemether-Lumefantrine Therapies and Selection of Pfcrt and Pfmdr1 Alleles in Nanoro, Burkina Faso
    Article Snippet: Briefly, an initial amplification of the outer region of each gene was followed by nested PCR using specific primers. .. For Pfmdr1 the following restriction enzymes were used: Afl III (NEB), Dra I (NEB), Dde I (NEB), Ase I (NEB), and Eco R V (NEB) respectively for Pfmdr1-N86Y , Pfmdr1 -Y184F, Pfmdr1 -S1034C, Pfmdr1 - N1042D and, Pfmdr1 -D1246Y.

    Article Title: Asymptomatic malaria infections and Pfmdr1 mutations in an endemic area of Nigeria
    Article Snippet: .. Nested PCR products were restricted by enzymatic digest with specific restriction enzymes: Afl III (New England Biolabs, USA) was used to detect mutation causing base change from asparagine (N) to tyrosine (Y) at codon 86 while EcoRV (Beijing TransGen Biotech, China) was used to detect mutation causing base change from aspartic acid (D) to tyrosine (N) at codon 1246. ..

    Chloramphenicol Acetyltransferase Assay:

    Article Title: Extraction of amplifiable DNA from embalmed human cadaver tissue
    Article Snippet: .. Amplified DNA was digested with HaeII and AflIII (NEB catR0107S and R0541S) for 2 h at 37 °C and resolved on a 4% agarose gel. ..

    Plasmid Preparation:

    Article Title: IMAGEtags: Quantifying mRNA transcription in real time with multiaptamer reporters
    Article Snippet: .. Digest pZErO-2 vector with Afl III (New England Biolabs-NEB, Ipswich, MA, Cat# R0541S) and Apo I (NEB, R0566S) restriction enzymes and isolate the 1854 base pair fragment using gel electrophoresis followed by extraction and purification by a method such as the QIAquick gel extraction kit (QIAGEN, Valencia, CA, Cat# 28704). .. Use two pairs of complementary oligonucleotides to create inserts containing between two and seven repeats (150–250bp) of the sequences flanked by restriction sites for Bsa I (NEB, R0535S) and Bsm AI (NEB, R0529S) for subsequent multiplication ( ).

    Article Title: Designing and overproducing a tandem epitope of gp350/220 that shows a potential to become an EBV vaccine
    Article Snippet: The sequence encoding the tandem epitope was synthesized by Genescript and inserted into pMAL-p5x plasmid. .. The insertion results were checked for validity based on the excision of the restriction enzyme (AflIII; NEB Cat No. R0541S).

    Software:

    Article Title: Asymptomatic malaria infections and Pfmdr1 mutations in an endemic area of Nigeria
    Article Snippet: Nested PCR products were restricted by enzymatic digest with specific restriction enzymes: Afl III (New England Biolabs, USA) was used to detect mutation causing base change from asparagine (N) to tyrosine (Y) at codon 86 while EcoRV (Beijing TransGen Biotech, China) was used to detect mutation causing base change from aspartic acid (D) to tyrosine (N) at codon 1246. .. Sequence alignment with reference 3D7 strain (PF3D7_0523000) was carried out using Geneious software® version 11.6.

    Selection:

    Article Title: Designing and overproducing a tandem epitope of gp350/220 that shows a potential to become an EBV vaccine
    Article Snippet: The insertion results were checked for validity based on the excision of the restriction enzyme (AflIII; NEB Cat No. R0541S). .. Next, selection on the successful transformation was performed by growing E. coli transformant on LB media enriched with ampicillin 100 ug/ml for overnight at 37 °C.

    Agarose Gel Electrophoresis:

    Article Title: Erratum to: ‘The Memory Aid study: protocol for a randomized controlled clinical trial evaluating the effect of computer-based working memory training in elderly patients with mild cognitive impairment (MCI)’
    Article Snippet: Then 15 μl of the amplification PCR products will be digested directly by 2.5 U of restriction enzymes MslI (R0571S, New England Biolabs, Beverly, MA) for 2 hours at 37 °C, and by Hae II (R0107S, New England Biolabs, Beverly, MA, USA) and Afl III (R0541S, New England Biolabs, Beverly, MA, USA) overnight at 37 °C. .. The digested PCR products will then be analyzed on 4 % agarose gel and visualized using GelGreen™ Nucleic Acid Gel Stain (89139–144, Biotium, Hayward, CA).

    Article Title: Light-dependent regulation of sleep/wake states by prokineticin 2 in zebrafish
    Article Snippet: PCR products were digested with AflIII (R0541, New England Biolabs Inc.). .. Mutant (527 bp) and WT (218 bp + 316 bp) bands were distinguished by running the digested PCR reaction on a 2% agarose gel.

    Article Title: Extraction of amplifiable DNA from embalmed human cadaver tissue
    Article Snippet: .. Amplified DNA was digested with HaeII and AflIII (NEB cat#R0107S and R0541S) for 2 h at 37 °C and resolved on a 4% agarose gel. ..

    Article Title: Correlation of Molecular Markers, Pfmdr1-N86Y and Pfcrt-K76T, with In Vitro Chloroquine Resistant Plasmodium falciparum, Isolated in the Malaria Endemic States of Assam and Arunachal Pradesh, Northeast India
    Article Snippet: Restriction Digestion with ApoI and Afl III The finally amplified product was subjected to restriction digestion with Afl III (mutational allele) and Apo I (wild type allele) (New England Biolabs, UK) by incubating at 37°C for one hour with the one unit of each enzyme. .. The digests were resolved on 3% agarose gel, stained with ethidium bromide, and results were recorded on the gel documentation system (UVITEC, UK).

    Staining:

    Article Title: Erratum to: ‘The Memory Aid study: protocol for a randomized controlled clinical trial evaluating the effect of computer-based working memory training in elderly patients with mild cognitive impairment (MCI)’
    Article Snippet: Then 15 μl of the amplification PCR products will be digested directly by 2.5 U of restriction enzymes MslI (R0571S, New England Biolabs, Beverly, MA) for 2 hours at 37 °C, and by Hae II (R0107S, New England Biolabs, Beverly, MA, USA) and Afl III (R0541S, New England Biolabs, Beverly, MA, USA) overnight at 37 °C. .. The digested PCR products will then be analyzed on 4 % agarose gel and visualized using GelGreen™ Nucleic Acid Gel Stain (89139–144, Biotium, Hayward, CA).

    Article Title: Correlation of Molecular Markers, Pfmdr1-N86Y and Pfcrt-K76T, with In Vitro Chloroquine Resistant Plasmodium falciparum, Isolated in the Malaria Endemic States of Assam and Arunachal Pradesh, Northeast India
    Article Snippet: Restriction Digestion with ApoI and Afl III The finally amplified product was subjected to restriction digestion with Afl III (mutational allele) and Apo I (wild type allele) (New England Biolabs, UK) by incubating at 37°C for one hour with the one unit of each enzyme. .. The digests were resolved on 3% agarose gel, stained with ethidium bromide, and results were recorded on the gel documentation system (UVITEC, UK).

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    New England Biolabs afl iii
    Flow diagram of RCA in situ . Pretreatment prior to in situ RCA detection of the Tp53 gene. Restriction enzymes were used to cut ≈20 base pairs either 3′ or 5′ of the probe binding site. <t>Afl</t> <t>III</t> was used for digestion 5′ of the binding site, and Bbs I was used for digestion 3′ of the target. Cells were then treated with exonuclease III, which digests DNA 3′ → 5′ starting with 3′ hydroxyl left by the endonuclease, resulting in staggered single-stranded DNA. The DNA strand remaining following Afl III digestion in this case is the complement to the sense probe sequence. DNA remaining following Bbs I digestion is the complement to the antisense probe sequence. Bbs I digestion constitutes a negative control for the RCA process using the sense probe, and Afl III constitutes a negative control for the antisense probe. The two ends of the sense probe create an incomplete circle as they anneal to the complementary site on the DNA digested with Bbs I. The DNA strand digested with Afl III is complementary to the sense probe and allows it to anneal and ligate, completing the circle and locking the probe onto the target. Targets other than Tp53 may require different endonucleases.
    Afl Iii, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Flow diagram of RCA in situ . Pretreatment prior to in situ RCA detection of the Tp53 gene. Restriction enzymes were used to cut ≈20 base pairs either 3′ or 5′ of the probe binding site. Afl III was used for digestion 5′ of the binding site, and Bbs I was used for digestion 3′ of the target. Cells were then treated with exonuclease III, which digests DNA 3′ → 5′ starting with 3′ hydroxyl left by the endonuclease, resulting in staggered single-stranded DNA. The DNA strand remaining following Afl III digestion in this case is the complement to the sense probe sequence. DNA remaining following Bbs I digestion is the complement to the antisense probe sequence. Bbs I digestion constitutes a negative control for the RCA process using the sense probe, and Afl III constitutes a negative control for the antisense probe. The two ends of the sense probe create an incomplete circle as they anneal to the complementary site on the DNA digested with Bbs I. The DNA strand digested with Afl III is complementary to the sense probe and allows it to anneal and ligate, completing the circle and locking the probe onto the target. Targets other than Tp53 may require different endonucleases.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Detection of DNA point mutations and mRNA expression levels by rolling circle amplification in individual cells

    doi: 10.1073/pnas.251383598

    Figure Lengend Snippet: Flow diagram of RCA in situ . Pretreatment prior to in situ RCA detection of the Tp53 gene. Restriction enzymes were used to cut ≈20 base pairs either 3′ or 5′ of the probe binding site. Afl III was used for digestion 5′ of the binding site, and Bbs I was used for digestion 3′ of the target. Cells were then treated with exonuclease III, which digests DNA 3′ → 5′ starting with 3′ hydroxyl left by the endonuclease, resulting in staggered single-stranded DNA. The DNA strand remaining following Afl III digestion in this case is the complement to the sense probe sequence. DNA remaining following Bbs I digestion is the complement to the antisense probe sequence. Bbs I digestion constitutes a negative control for the RCA process using the sense probe, and Afl III constitutes a negative control for the antisense probe. The two ends of the sense probe create an incomplete circle as they anneal to the complementary site on the DNA digested with Bbs I. The DNA strand digested with Afl III is complementary to the sense probe and allows it to anneal and ligate, completing the circle and locking the probe onto the target. Targets other than Tp53 may require different endonucleases.

    Article Snippet: Either Afl III or Bbs I (0.1 unit/μl, New England Biolabs) was applied for 12 h at 37°C.

    Techniques: Flow Cytometry, In Situ, Binding Assay, Sequencing, Negative Control

    Map of PCR primer binding sites and restriction enzyme cut sizes for the barcode fragment of COI. BamHI restriction sites are only found in haplotype group A alleles while AflIII and BseYI restriction sites are only found in haplotype group B alleles.

    Journal: Systematic entomology

    Article Title: A tale of two haplotype groups: Evaluating the New World Junonia ring species hypothesis using the distribution of divergent COI haplotypes

    doi: 10.1111/syen.12120

    Figure Lengend Snippet: Map of PCR primer binding sites and restriction enzyme cut sizes for the barcode fragment of COI. BamHI restriction sites are only found in haplotype group A alleles while AflIII and BseYI restriction sites are only found in haplotype group B alleles.

    Article Snippet: PCR products of the correct size were evaluated by a triple digest using BseYI, AflIII, and BamHI restriction endonucleases (New England Biolabs, Ipswich, MA, USA) ( ).

    Techniques: Polymerase Chain Reaction, Binding Assay

    Examples of Southern blot results. Genotyping using FMR1 probe: After EcoRI /EagI double digestion, normal females exhibit two fragments, a ~2.8 kb (active X) and a ~5.2 kb (inactive X), while in normal males only a ~2.8 kb fragment is seen: (a) FXS male mosaic for a 51 CGG allele and a full mutation; (b) Premutated FMR1 male (110 CGGs); (c) Klinefelter with two FMR1 normal sized-alleles, showing the active and the inactive X-chromosome; (d) Normal FMR1 male (19 CGGs); (e) Premutated FMR1 male (58 CGGs). Genotyping using AFF2 probe: Following AflIII /NotI double digestion, normal females present two fragments, a ~2.2 kb (active X) and a ~4.8 kb (inactive X), whereas normal males exhibit only a ~2.2 kb fragment: (f) Intermediate AFF2 male (47 CCGs); (g) Premutated AFF2 male (68 CCGs); (h) Homoallelic female [(CCG) 14 /(CCG) 14 ].

    Journal: BMC Medical Genetics

    Article Title: Development and validation of a multiplex-PCR assay for X-linked intellectual disability

    doi: 10.1186/1471-2350-14-80

    Figure Lengend Snippet: Examples of Southern blot results. Genotyping using FMR1 probe: After EcoRI /EagI double digestion, normal females exhibit two fragments, a ~2.8 kb (active X) and a ~5.2 kb (inactive X), while in normal males only a ~2.8 kb fragment is seen: (a) FXS male mosaic for a 51 CGG allele and a full mutation; (b) Premutated FMR1 male (110 CGGs); (c) Klinefelter with two FMR1 normal sized-alleles, showing the active and the inactive X-chromosome; (d) Normal FMR1 male (19 CGGs); (e) Premutated FMR1 male (58 CGGs). Genotyping using AFF2 probe: Following AflIII /NotI double digestion, normal females present two fragments, a ~2.2 kb (active X) and a ~4.8 kb (inactive X), whereas normal males exhibit only a ~2.2 kb fragment: (f) Intermediate AFF2 male (47 CCGs); (g) Premutated AFF2 male (68 CCGs); (h) Homoallelic female [(CCG) 14 /(CCG) 14 ].

    Article Snippet: Southern blot genotyping was done using ~10 μg of gDNA double-digested with the restriction enzymes EcoRI and EagI for FMR1 gene or AflIII and NotI for AFF2 gene (New England Biolabs, Ipswich, MA, USA).

    Techniques: Southern Blot, Mutagenesis

    Representative photomicrograph showing the results of RFLP after digestion of PCR amplified product with endonucleases, Apo1 and AfI III for Pfmdr1 gene (86Y) in chloroquine resistant Plasmodium falciparum isolates. In photograph, (M) is 100 bp DNA ladder; (C) is undigested product as control; (P) is Apo1; (F) is Afl III and (R1- R4) are chloroquine resistant P. falciparum isolates.

    Journal: PLoS ONE

    Article Title: Correlation of Molecular Markers, Pfmdr1-N86Y and Pfcrt-K76T, with In Vitro Chloroquine Resistant Plasmodium falciparum, Isolated in the Malaria Endemic States of Assam and Arunachal Pradesh, Northeast India

    doi: 10.1371/journal.pone.0103848

    Figure Lengend Snippet: Representative photomicrograph showing the results of RFLP after digestion of PCR amplified product with endonucleases, Apo1 and AfI III for Pfmdr1 gene (86Y) in chloroquine resistant Plasmodium falciparum isolates. In photograph, (M) is 100 bp DNA ladder; (C) is undigested product as control; (P) is Apo1; (F) is Afl III and (R1- R4) are chloroquine resistant P. falciparum isolates.

    Article Snippet: Restriction Digestion with ApoI and Afl III The finally amplified product was subjected to restriction digestion with Afl III (mutational allele) and Apo I (wild type allele) (New England Biolabs, UK) by incubating at 37°C for one hour with the one unit of each enzyme.

    Techniques: Polymerase Chain Reaction, Amplification

    Representative photomicrograph showing the results of RFLP after digestion of PCR amplified product with endonucleases, Apo1 and AfI III for Pfmdr1 gene (N86) in chloroquine sensitive Plasmodium falciparum isolates. In photograph, (M) is 100 bp DNA ladder; (C) is undigested product as control; (P) is Apo1; (F) is Afl III and (ST1-ST4) are chloroquine sensitive P. falciparum isolates.

    Journal: PLoS ONE

    Article Title: Correlation of Molecular Markers, Pfmdr1-N86Y and Pfcrt-K76T, with In Vitro Chloroquine Resistant Plasmodium falciparum, Isolated in the Malaria Endemic States of Assam and Arunachal Pradesh, Northeast India

    doi: 10.1371/journal.pone.0103848

    Figure Lengend Snippet: Representative photomicrograph showing the results of RFLP after digestion of PCR amplified product with endonucleases, Apo1 and AfI III for Pfmdr1 gene (N86) in chloroquine sensitive Plasmodium falciparum isolates. In photograph, (M) is 100 bp DNA ladder; (C) is undigested product as control; (P) is Apo1; (F) is Afl III and (ST1-ST4) are chloroquine sensitive P. falciparum isolates.

    Article Snippet: Restriction Digestion with ApoI and Afl III The finally amplified product was subjected to restriction digestion with Afl III (mutational allele) and Apo I (wild type allele) (New England Biolabs, UK) by incubating at 37°C for one hour with the one unit of each enzyme.

    Techniques: Polymerase Chain Reaction, Amplification