polymerase chain reaction restriction fragment length polymorphism pcr rflp  (New England Biolabs)


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    Name:
    Poly U Polymerase
    Description:
    Poly U Polymerase 60 units
    Catalog Number:
    m0337s
    Price:
    90
    Size:
    60 units
    Category:
    RNA Polymerases
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    New England Biolabs polymerase chain reaction restriction fragment length polymorphism pcr rflp
    Poly U Polymerase
    Poly U Polymerase 60 units
    https://www.bioz.com/result/polymerase chain reaction restriction fragment length polymorphism pcr rflp/product/New England Biolabs
    Average 99 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    polymerase chain reaction restriction fragment length polymorphism pcr rflp - by Bioz Stars, 2020-08
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    1) Product Images from "A novel connexin 50 (GJA8) mutation in a Chinese family with a dominant congenital pulverulent nuclear cataract"

    Article Title: A novel connexin 50 (GJA8) mutation in a Chinese family with a dominant congenital pulverulent nuclear cataract

    Journal: Molecular Vision

    doi:

    Mutation analysis of GJA8 . Sequence chromatograms of the wild-type allele ( A ) demonstrate a nucleotide sequence encoding a serine (Ser) at codon 276, and the chromatograms of the mutant allele ( B ) demonstrate a C to T transition resulting in the substitution of serine (Ser) by phenylalanine (Phe). Confirmation of segregation of the S276F mutation was given by the PCR-RFLP method ( C ). M is the DNA ladder; Lane 1 shows the digestions of the PCR products amplified from samples of the patients by Hpy188I. Lane 2 and 4 show the PCR products amplified from samples of one patient or the unaffected member in the pedigree. Lane 3 illustrates the digestions of the PCR products amplified from samples of the unaffected family member by Hpy188I. The mutant primer results in the gain of Hpy 188I sites producing digested fragments of 215 and 24 bp with wild-type GJA8 alleles, and the mutation in the GJA8 gene leads to an abolition of this site, remaining undigested (239 bp).
    Figure Legend Snippet: Mutation analysis of GJA8 . Sequence chromatograms of the wild-type allele ( A ) demonstrate a nucleotide sequence encoding a serine (Ser) at codon 276, and the chromatograms of the mutant allele ( B ) demonstrate a C to T transition resulting in the substitution of serine (Ser) by phenylalanine (Phe). Confirmation of segregation of the S276F mutation was given by the PCR-RFLP method ( C ). M is the DNA ladder; Lane 1 shows the digestions of the PCR products amplified from samples of the patients by Hpy188I. Lane 2 and 4 show the PCR products amplified from samples of one patient or the unaffected member in the pedigree. Lane 3 illustrates the digestions of the PCR products amplified from samples of the unaffected family member by Hpy188I. The mutant primer results in the gain of Hpy 188I sites producing digested fragments of 215 and 24 bp with wild-type GJA8 alleles, and the mutation in the GJA8 gene leads to an abolition of this site, remaining undigested (239 bp).

    Techniques Used: Mutagenesis, Sequencing, Polymerase Chain Reaction, Amplification

    Related Articles

    Amplification:

    Article Title: Circularization pathway of a bacterial group II intron
    Article Snippet: .. For Ll.LtrB-ΔA a second amplification of the 3′ end was performed where a polyU instead of a polyA tail was added using 2U of Poly(U) Polymerase (New England Biolabs) (10 min at 37°C). ..

    Article Title: Serotype-specific interactions among functional domains of dengue virus 2 nonstructural proteins (NS) 5 and NS3 are crucial for viral RNA replication
    Article Snippet: .. The synthesized viral cDNA from collected supernatants was amplified by PCR using NEBNext High Fidelity 2× polymerase (New England Biolabs) and the viral sequence-specific primers (Ref. and ) to make overlapping fragments (each ∼800 bp) that spanned the entire genome. .. PCR products were purified by agarose gel (Zymo Research), and the nucleotide sequence of each fragment was determined by GENEWIZ Inc.

    In Vitro:

    Article Title: An Unusual Two-Step Control of CPEB Destruction by Pin1
    Article Snippet: .. Fifty oocytes [some injected with 10 ng of mRNA synthesized with an Ambion mMESSAGE kit and polyadenylated in vitro with Escherichia coli poly(A) polymerase (New England BioLabs)] or HEK293T cells from one 10-cm dish were homogenized in lysis buffer containing 150 mM NaCl, 1% NP-40, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris-HCl (pH 7.7), 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM dithiothreitol, and protease inhibitor cocktail (Roche). .. The lysis buffer in some experiments also contained 10 μg/ml RNase A (Sigma).

    Synthesized:

    Article Title: Serotype-specific interactions among functional domains of dengue virus 2 nonstructural proteins (NS) 5 and NS3 are crucial for viral RNA replication
    Article Snippet: .. The synthesized viral cDNA from collected supernatants was amplified by PCR using NEBNext High Fidelity 2× polymerase (New England Biolabs) and the viral sequence-specific primers (Ref. and ) to make overlapping fragments (each ∼800 bp) that spanned the entire genome. .. PCR products were purified by agarose gel (Zymo Research), and the nucleotide sequence of each fragment was determined by GENEWIZ Inc.

    Article Title: An Unusual Two-Step Control of CPEB Destruction by Pin1
    Article Snippet: .. Fifty oocytes [some injected with 10 ng of mRNA synthesized with an Ambion mMESSAGE kit and polyadenylated in vitro with Escherichia coli poly(A) polymerase (New England BioLabs)] or HEK293T cells from one 10-cm dish were homogenized in lysis buffer containing 150 mM NaCl, 1% NP-40, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris-HCl (pH 7.7), 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM dithiothreitol, and protease inhibitor cocktail (Roche). .. The lysis buffer in some experiments also contained 10 μg/ml RNase A (Sigma).

    Mutagenesis:

    Article Title: Sequences in the U3 region of Human Immunodeficiency Virus 1 improve efficiency of minus strand transfer in infected cells
    Article Snippet: .. For mutant M9-m, two PCR products were produced using pairs of oligomers 1/3 and 4/2 and VENT polymerase (NewEngland Biolabs). ..

    Protease Inhibitor:

    Article Title: An Unusual Two-Step Control of CPEB Destruction by Pin1
    Article Snippet: .. Fifty oocytes [some injected with 10 ng of mRNA synthesized with an Ambion mMESSAGE kit and polyadenylated in vitro with Escherichia coli poly(A) polymerase (New England BioLabs)] or HEK293T cells from one 10-cm dish were homogenized in lysis buffer containing 150 mM NaCl, 1% NP-40, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris-HCl (pH 7.7), 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM dithiothreitol, and protease inhibitor cocktail (Roche). .. The lysis buffer in some experiments also contained 10 μg/ml RNase A (Sigma).

    Quantitation Assay:

    Article Title: Decreased miR-155-5p, miR-15a, and miR-186 Expression in Gastric Cancer Is Associated with Advanced Tumor Grade and Metastasis
    Article Snippet: .. MiRNA quantitation Briefly, a poly-A tail was added to total RNAs (500 ng) using Poly-A polymerase (New England Biolabs, UK) at 37 °C for 30 min according to manufacturer’s protocol. .. For cDNA synthesis, we used PrimeScript™ 1st strand cDNA Synthesis Kit (TaKaRa, Japan) and an RT adaptor primer ( ).

    Produced:

    Article Title: Sequences in the U3 region of Human Immunodeficiency Virus 1 improve efficiency of minus strand transfer in infected cells
    Article Snippet: .. For mutant M9-m, two PCR products were produced using pairs of oligomers 1/3 and 4/2 and VENT polymerase (NewEngland Biolabs). ..

    Polymerase Chain Reaction:

    Article Title: Serotype-specific interactions among functional domains of dengue virus 2 nonstructural proteins (NS) 5 and NS3 are crucial for viral RNA replication
    Article Snippet: .. The synthesized viral cDNA from collected supernatants was amplified by PCR using NEBNext High Fidelity 2× polymerase (New England Biolabs) and the viral sequence-specific primers (Ref. and ) to make overlapping fragments (each ∼800 bp) that spanned the entire genome. .. PCR products were purified by agarose gel (Zymo Research), and the nucleotide sequence of each fragment was determined by GENEWIZ Inc.

    Article Title: Sequences in the U3 region of Human Immunodeficiency Virus 1 improve efficiency of minus strand transfer in infected cells
    Article Snippet: .. For mutant M9-m, two PCR products were produced using pairs of oligomers 1/3 and 4/2 and VENT polymerase (NewEngland Biolabs). ..

    Incubation:

    Article Title: Hallmarks of Hepatitis C Virus in Equine Hepacivirus
    Article Snippet: .. The poly(U) tail was added to the 3′ end of the RNA preparation using Escherichia coli poly(U) polymerase (New England BioLabs, Ipswich, MA) and was incubated for 45 min at 37°C. .. The resulting preparation was reverse transcribed by the SuperScript First-Strand Synthesis system (Life Technologies) using an oligo(dA) adapter primer (5′-TTGCGAGCACAGAATTAATACGACTCACAAAAAAAAAAAAVN-3′).

    Sequencing:

    Article Title: Serotype-specific interactions among functional domains of dengue virus 2 nonstructural proteins (NS) 5 and NS3 are crucial for viral RNA replication
    Article Snippet: .. The synthesized viral cDNA from collected supernatants was amplified by PCR using NEBNext High Fidelity 2× polymerase (New England Biolabs) and the viral sequence-specific primers (Ref. and ) to make overlapping fragments (each ∼800 bp) that spanned the entire genome. .. PCR products were purified by agarose gel (Zymo Research), and the nucleotide sequence of each fragment was determined by GENEWIZ Inc.

    Lysis:

    Article Title: An Unusual Two-Step Control of CPEB Destruction by Pin1
    Article Snippet: .. Fifty oocytes [some injected with 10 ng of mRNA synthesized with an Ambion mMESSAGE kit and polyadenylated in vitro with Escherichia coli poly(A) polymerase (New England BioLabs)] or HEK293T cells from one 10-cm dish were homogenized in lysis buffer containing 150 mM NaCl, 1% NP-40, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris-HCl (pH 7.7), 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM dithiothreitol, and protease inhibitor cocktail (Roche). .. The lysis buffer in some experiments also contained 10 μg/ml RNase A (Sigma).

    Injection:

    Article Title: An Unusual Two-Step Control of CPEB Destruction by Pin1
    Article Snippet: .. Fifty oocytes [some injected with 10 ng of mRNA synthesized with an Ambion mMESSAGE kit and polyadenylated in vitro with Escherichia coli poly(A) polymerase (New England BioLabs)] or HEK293T cells from one 10-cm dish were homogenized in lysis buffer containing 150 mM NaCl, 1% NP-40, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris-HCl (pH 7.7), 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM dithiothreitol, and protease inhibitor cocktail (Roche). .. The lysis buffer in some experiments also contained 10 μg/ml RNase A (Sigma).

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    New England Biolabs tcsc5d amplification
    Key polymorphic sites in the T. cruzi <t>TcSC5D</t> gene. A: diploid genotypes built from identified highly informative sites (see main text), showing a section of one chromatogram around the tetra-allelic SNP at position 657. B: Summary of observed nucleotide changes between DTUs for these six sites. In the case of the comparisons with the TcII DTU, this is the minimum expected number of changes (marked with * in the figure), because of the diversity observed at position 618 in strains from this DTU.
    Tcsc5d Amplification, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs rflp
    The <t>ARMS-PCR</t> and <t>RFLP</t> established in this study for the genotyping at positions +401 (A), +408 (B) and +428 (C). The primers and PCR conditions are listed in Table 1 . The PCR products (A and C) and the RFLP products (B) were resolved in 2% agarose gel and photographed.
    Rflp, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 74 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs restriction fragment length polymorphism rflp analysis
    Detection of mutations in codon 13 of Ki- ras in colorectal cell lines. <t>RFLP</t> analysis was performed on Ki- ras PCR <t>amplicons</t> with the restriction enzyme Bsl I. Mutations in the first or second base of codon 13 will abolish the Bsl I site; therefore Ki- ras codon 13 mutants are resistant to digestion. Wild-type (WT) amplicons, and mutants other than codon 13, are fully digested by Bsl I. Undigested amplicons are shown in the last lane. Both LIM1215 and RKO have WT Ki- ras ; LS174T is a codon 12 mutant; HCT116, displaying undigested product, is a codon 13 mutant. MW is pUC19/ Msp I.
    Restriction Fragment Length Polymorphism Rflp Analysis, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 92/100, based on 41 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs polymerase chain reaction restriction fragment length polymorphism pcr rflp
    Mutation analysis of GJA8 . Sequence chromatograms of the wild-type allele ( A ) demonstrate a nucleotide sequence encoding a serine (Ser) at codon 276, and the chromatograms of the mutant allele ( B ) demonstrate a C to T transition resulting in the substitution of serine (Ser) by phenylalanine (Phe). Confirmation of segregation of the S276F mutation was given by the <t>PCR-RFLP</t> method ( C ). M is the DNA ladder; Lane 1 shows the digestions of the PCR products amplified from samples of the patients by Hpy188I. Lane 2 and 4 show the PCR products amplified from samples of one patient or the unaffected member in the pedigree. Lane 3 illustrates the digestions of the PCR products amplified from samples of the unaffected family member by Hpy188I. The mutant primer results in the gain of Hpy 188I sites producing digested fragments of 215 and 24 bp with wild-type GJA8 alleles, and the mutation in the GJA8 gene leads to an abolition of this site, remaining undigested (239 bp).
    Polymerase Chain Reaction Restriction Fragment Length Polymorphism Pcr Rflp, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Key polymorphic sites in the T. cruzi TcSC5D gene. A: diploid genotypes built from identified highly informative sites (see main text), showing a section of one chromatogram around the tetra-allelic SNP at position 657. B: Summary of observed nucleotide changes between DTUs for these six sites. In the case of the comparisons with the TcII DTU, this is the minimum expected number of changes (marked with * in the figure), because of the diversity observed at position 618 in strains from this DTU.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: A Simple Strain Typing Assay for Trypanosoma cruzi: Discrimination of Major Evolutionary Lineages from a Single Amplification Product

    doi: 10.1371/journal.pntd.0001777

    Figure Lengend Snippet: Key polymorphic sites in the T. cruzi TcSC5D gene. A: diploid genotypes built from identified highly informative sites (see main text), showing a section of one chromatogram around the tetra-allelic SNP at position 657. B: Summary of observed nucleotide changes between DTUs for these six sites. In the case of the comparisons with the TcII DTU, this is the minimum expected number of changes (marked with * in the figure), because of the diversity observed at position 618 in strains from this DTU.

    Article Snippet: An aliquot (20 µl) of the TcSC5D amplification products where digested in a single incubation with 1 U of Hpa I (NEB R0105) and 1 U Sph I (NEB R0182) at 37 for 1 h. Digestion was performed in the same buffer used for PCR amplification (Invitrogen Taq PCR Buffer).

    Techniques:

    The TcSC5D locus as a lineage discriminant marker. A: schematic view of the TcSC5D amplicon, with all identified SNPs, including key discriminant positions (marked in blue), and polymorphic HpaI/SphI restriction enzyme sites, showing their presence/absence in each lineage. B: Restriction fragment length polymorphism analysis of the TcSC5D amplification product. Fragments of the SphI/HpaI double digestion were resolved in a 2% TBE-agarose gel. Lanes in the gel correspond to: molecular size markers (lane 1), and DNA from T. cruzi strains (lanes 2–21). These are: Sylvio X10 (lane 3), Dm28c (4), and CAI72 (5) for DTU TcI; MAS1 cl1 (6), TU18 cl93 (7), and IVV cl4 (8) for DTU TcII; M6241 cl6 (9), M5631 cl5 (10), and X109/2 (11) for DUT TcIII; CanIII cl1(12), Dog Theis (13) and 92122102R (14) for DTU TcIV; Sc43 (15), MN cl2 (16) and Teh53 (17) for DTU TcV; CL-Brener (18), P63 cl1 (19), and Tulahuen cl2 (20) for DTU TcVI; and TCC1994 and TCC1122 for DTU Tcbat. The corresponding locus from T. cruzi marinkellei was analyzed in lanes 22/23 (stocks B3 and B7).

    Journal: PLoS Neglected Tropical Diseases

    Article Title: A Simple Strain Typing Assay for Trypanosoma cruzi: Discrimination of Major Evolutionary Lineages from a Single Amplification Product

    doi: 10.1371/journal.pntd.0001777

    Figure Lengend Snippet: The TcSC5D locus as a lineage discriminant marker. A: schematic view of the TcSC5D amplicon, with all identified SNPs, including key discriminant positions (marked in blue), and polymorphic HpaI/SphI restriction enzyme sites, showing their presence/absence in each lineage. B: Restriction fragment length polymorphism analysis of the TcSC5D amplification product. Fragments of the SphI/HpaI double digestion were resolved in a 2% TBE-agarose gel. Lanes in the gel correspond to: molecular size markers (lane 1), and DNA from T. cruzi strains (lanes 2–21). These are: Sylvio X10 (lane 3), Dm28c (4), and CAI72 (5) for DTU TcI; MAS1 cl1 (6), TU18 cl93 (7), and IVV cl4 (8) for DTU TcII; M6241 cl6 (9), M5631 cl5 (10), and X109/2 (11) for DUT TcIII; CanIII cl1(12), Dog Theis (13) and 92122102R (14) for DTU TcIV; Sc43 (15), MN cl2 (16) and Teh53 (17) for DTU TcV; CL-Brener (18), P63 cl1 (19), and Tulahuen cl2 (20) for DTU TcVI; and TCC1994 and TCC1122 for DTU Tcbat. The corresponding locus from T. cruzi marinkellei was analyzed in lanes 22/23 (stocks B3 and B7).

    Article Snippet: An aliquot (20 µl) of the TcSC5D amplification products where digested in a single incubation with 1 U of Hpa I (NEB R0105) and 1 U Sph I (NEB R0182) at 37 for 1 h. Digestion was performed in the same buffer used for PCR amplification (Invitrogen Taq PCR Buffer).

    Techniques: Marker, Amplification, Agarose Gel Electrophoresis

    Proposed typing strategy based on the TcSC5D locus. A : the highly streamlined TcSC5D-PCR-RFLP assay can discriminate all non-hybrid lineages. If discrimination of DTUs TcV from TcVI is necessary, sequencing of the TcSC5D amplification locus is required. B : alternatively, if the method of choice is the PCR-RFLP, then a second locus can be assayed to resolve these DTUs. This additional locus can be the TcMK gene as shown in the figure (this work, see main text), or the gp72 gene (polymorphic Taq I site [41] ).

    Journal: PLoS Neglected Tropical Diseases

    Article Title: A Simple Strain Typing Assay for Trypanosoma cruzi: Discrimination of Major Evolutionary Lineages from a Single Amplification Product

    doi: 10.1371/journal.pntd.0001777

    Figure Lengend Snippet: Proposed typing strategy based on the TcSC5D locus. A : the highly streamlined TcSC5D-PCR-RFLP assay can discriminate all non-hybrid lineages. If discrimination of DTUs TcV from TcVI is necessary, sequencing of the TcSC5D amplification locus is required. B : alternatively, if the method of choice is the PCR-RFLP, then a second locus can be assayed to resolve these DTUs. This additional locus can be the TcMK gene as shown in the figure (this work, see main text), or the gp72 gene (polymorphic Taq I site [41] ).

    Article Snippet: An aliquot (20 µl) of the TcSC5D amplification products where digested in a single incubation with 1 U of Hpa I (NEB R0105) and 1 U Sph I (NEB R0182) at 37 for 1 h. Digestion was performed in the same buffer used for PCR amplification (Invitrogen Taq PCR Buffer).

    Techniques: Polymerase Chain Reaction, RFLP Assay, Sequencing, Amplification

    The ARMS-PCR and RFLP established in this study for the genotyping at positions +401 (A), +408 (B) and +428 (C). The primers and PCR conditions are listed in Table 1 . The PCR products (A and C) and the RFLP products (B) were resolved in 2% agarose gel and photographed.

    Journal: Veterinary Research

    Article Title: Identification and genotyping of feline infectious peritonitis-associated single nucleotide polymorphisms in the feline interferon-? gene

    doi: 10.1186/1297-9716-45-57

    Figure Lengend Snippet: The ARMS-PCR and RFLP established in this study for the genotyping at positions +401 (A), +408 (B) and +428 (C). The primers and PCR conditions are listed in Table 1 . The PCR products (A and C) and the RFLP products (B) were resolved in 2% agarose gel and photographed.

    Article Snippet: The PCR for the genotyping of SNP with RFLP was carried out with the same protocol, and the PCR products were digested with HpyCH4 III (New England Biolabs, Ipswich, USA) following the manufacturer’s instructions.

    Techniques: Polymerase Chain Reaction, Agarose Gel Electrophoresis

    Detection of mutations in codon 13 of Ki- ras in colorectal cell lines. RFLP analysis was performed on Ki- ras PCR amplicons with the restriction enzyme Bsl I. Mutations in the first or second base of codon 13 will abolish the Bsl I site; therefore Ki- ras codon 13 mutants are resistant to digestion. Wild-type (WT) amplicons, and mutants other than codon 13, are fully digested by Bsl I. Undigested amplicons are shown in the last lane. Both LIM1215 and RKO have WT Ki- ras ; LS174T is a codon 12 mutant; HCT116, displaying undigested product, is a codon 13 mutant. MW is pUC19/ Msp I.

    Journal: British Journal of Cancer

    Article Title: CpG island methylation is a common finding in colorectal cancer cell lines

    doi: 10.1038/sj.bjc.6600699

    Figure Lengend Snippet: Detection of mutations in codon 13 of Ki- ras in colorectal cell lines. RFLP analysis was performed on Ki- ras PCR amplicons with the restriction enzyme Bsl I. Mutations in the first or second base of codon 13 will abolish the Bsl I site; therefore Ki- ras codon 13 mutants are resistant to digestion. Wild-type (WT) amplicons, and mutants other than codon 13, are fully digested by Bsl I. Undigested amplicons are shown in the last lane. Both LIM1215 and RKO have WT Ki- ras ; LS174T is a codon 12 mutant; HCT116, displaying undigested product, is a codon 13 mutant. MW is pUC19/ Msp I.

    Article Snippet: Restriction fragment length polymorphism (RFLP) analysis was performed on amplicons at 55°C for 16 h with 10 U of Bsl I (NEB) according to the manufacturer's instructions.

    Techniques: Polymerase Chain Reaction, Mutagenesis

    Mutation analysis of GJA8 . Sequence chromatograms of the wild-type allele ( A ) demonstrate a nucleotide sequence encoding a serine (Ser) at codon 276, and the chromatograms of the mutant allele ( B ) demonstrate a C to T transition resulting in the substitution of serine (Ser) by phenylalanine (Phe). Confirmation of segregation of the S276F mutation was given by the PCR-RFLP method ( C ). M is the DNA ladder; Lane 1 shows the digestions of the PCR products amplified from samples of the patients by Hpy188I. Lane 2 and 4 show the PCR products amplified from samples of one patient or the unaffected member in the pedigree. Lane 3 illustrates the digestions of the PCR products amplified from samples of the unaffected family member by Hpy188I. The mutant primer results in the gain of Hpy 188I sites producing digested fragments of 215 and 24 bp with wild-type GJA8 alleles, and the mutation in the GJA8 gene leads to an abolition of this site, remaining undigested (239 bp).

    Journal: Molecular Vision

    Article Title: A novel connexin 50 (GJA8) mutation in a Chinese family with a dominant congenital pulverulent nuclear cataract

    doi:

    Figure Lengend Snippet: Mutation analysis of GJA8 . Sequence chromatograms of the wild-type allele ( A ) demonstrate a nucleotide sequence encoding a serine (Ser) at codon 276, and the chromatograms of the mutant allele ( B ) demonstrate a C to T transition resulting in the substitution of serine (Ser) by phenylalanine (Phe). Confirmation of segregation of the S276F mutation was given by the PCR-RFLP method ( C ). M is the DNA ladder; Lane 1 shows the digestions of the PCR products amplified from samples of the patients by Hpy188I. Lane 2 and 4 show the PCR products amplified from samples of one patient or the unaffected member in the pedigree. Lane 3 illustrates the digestions of the PCR products amplified from samples of the unaffected family member by Hpy188I. The mutant primer results in the gain of Hpy 188I sites producing digested fragments of 215 and 24 bp with wild-type GJA8 alleles, and the mutation in the GJA8 gene leads to an abolition of this site, remaining undigested (239 bp).

    Article Snippet: Restriction analysis Segregation of the mutation in the other participating family members was determined by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method with the restriction enzyme Hpy188I (New England Biolab, Beijing, China).

    Techniques: Mutagenesis, Sequencing, Polymerase Chain Reaction, Amplification