tcsc5d amplification  (New England Biolabs)


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    New England Biolabs tcsc5d amplification
    SphI
    SphI 2 500 units
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    Average 85 stars, based on 427 article reviews
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    Images

    1) Product Images from "A Simple Strain Typing Assay for Trypanosoma cruzi: Discrimination of Major Evolutionary Lineages from a Single Amplification Product"

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

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0001777

    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.
    Figure Legend 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.

    Techniques Used:

    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).
    Figure Legend 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).

    Techniques Used: 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] ).
    Figure Legend 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] ).

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

    2) Product Images from "Generation of Replication-Competent Hepatitis B Virus Genome from Blood Samples for Functional Characterization"

    Article Title: Generation of Replication-Competent Hepatitis B Virus Genome from Blood Samples for Functional Characterization

    Journal: Methods in molecular biology (Clifton, N.J.)

    doi: 10.1007/978-1-4939-6700-1_18

    Flow chart for the generation of replication-competent HBV genomes from blood samples. Virion-associated HBV DNA has the minus strand DNA ( thick line ) complete. The sense (S) primer anneals to its 3’ end to generate full-length plus strand, which will serve as the template for the antisense (AS) primer to generate more minus strand DNA. The HindIII and SacI sites introduced to the sense and antisense primers, respectively, will allow efficient cloning of the PCR product to pUC18 vector, whereas the internal BspQI sites allow subsequent precise release of the HBV genome. Such a linear HBV genome can be ligated in vitro to make it replication competent (capable of producing the terminally redundant pg RNA), or the ligated DNA is further digested with SphI and ligated with SphI cut, dephosphorylated pUC18 DNA. Bacterial colonies harboring tandem SphI dimer can be screened by hybridization with an oligoprobe spanning the SphI site. The 3.5-kb pg RNA can be produced from such a tandem dimer construct
    Figure Legend Snippet: Flow chart for the generation of replication-competent HBV genomes from blood samples. Virion-associated HBV DNA has the minus strand DNA ( thick line ) complete. The sense (S) primer anneals to its 3’ end to generate full-length plus strand, which will serve as the template for the antisense (AS) primer to generate more minus strand DNA. The HindIII and SacI sites introduced to the sense and antisense primers, respectively, will allow efficient cloning of the PCR product to pUC18 vector, whereas the internal BspQI sites allow subsequent precise release of the HBV genome. Such a linear HBV genome can be ligated in vitro to make it replication competent (capable of producing the terminally redundant pg RNA), or the ligated DNA is further digested with SphI and ligated with SphI cut, dephosphorylated pUC18 DNA. Bacterial colonies harboring tandem SphI dimer can be screened by hybridization with an oligoprobe spanning the SphI site. The 3.5-kb pg RNA can be produced from such a tandem dimer construct

    Techniques Used: Flow Cytometry, Clone Assay, Polymerase Chain Reaction, Plasmid Preparation, In Vitro, Hybridization, Produced, Construct

    3) Product Images from "In Vivo Bypass of 8-oxodG"

    Article Title: In Vivo Bypass of 8-oxodG

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1003682

    Models for incorporation of oligo GO and UGO. (A) Incorporation of Oligo GO creates three mismatches; if the oligo is not removed before the second round, replication of the C, indicated in yellow, would result in a Trp+ phenotype, replication of the 8-oxoG, indicated in red, would create a Bfa I site if an A were incorporated, and replication of the G indicated in blue would create an Sph I site. In the first round, usually the entire portion of the oligo containing the marked bases remains or is removed by MMR. However, when MMR is present, the segment of the oligo containing the 5′-two mismatches can be removed, but the segment creating the Trp+ phenotype can be left, presumably due to failure of MMR to recognized the C-C mismatch (see text). If the marked segment of Oligo GO persists to the second round of replication, translesion synthesis creates an Sph I site and a Bfa I site if the 8-oxoG is bypassed with an A. If the 8-oxoG induces template switching, no Sph I site is created. (B) Incorporation of Oligo UGO creates an additional mismatch with the C indicated in blue, 3′ of the C-C mismatch. Due to this additional mismatch, in the first round, either the entire oligo remains, or is removed. The results of the second round of replication are identical to that of Oligo GO in (A). Cells with a gray background are Trp- and thus do not survive selection.
    Figure Legend Snippet: Models for incorporation of oligo GO and UGO. (A) Incorporation of Oligo GO creates three mismatches; if the oligo is not removed before the second round, replication of the C, indicated in yellow, would result in a Trp+ phenotype, replication of the 8-oxoG, indicated in red, would create a Bfa I site if an A were incorporated, and replication of the G indicated in blue would create an Sph I site. In the first round, usually the entire portion of the oligo containing the marked bases remains or is removed by MMR. However, when MMR is present, the segment of the oligo containing the 5′-two mismatches can be removed, but the segment creating the Trp+ phenotype can be left, presumably due to failure of MMR to recognized the C-C mismatch (see text). If the marked segment of Oligo GO persists to the second round of replication, translesion synthesis creates an Sph I site and a Bfa I site if the 8-oxoG is bypassed with an A. If the 8-oxoG induces template switching, no Sph I site is created. (B) Incorporation of Oligo UGO creates an additional mismatch with the C indicated in blue, 3′ of the C-C mismatch. Due to this additional mismatch, in the first round, either the entire oligo remains, or is removed. The results of the second round of replication are identical to that of Oligo GO in (A). Cells with a gray background are Trp- and thus do not survive selection.

    Techniques Used: Translesion Synthesis, Selection

    Template switching induced by 8-oxoG. Template switching was determined as those transformants that did not contain the Sph I restriction site introduced by the oligo. More than 40 Trp+ revertants were assayed for the presence of an Sph I site in each strain of the indicated genotype. Error bars represent standard deviation of the mean for cases in which three or more independent experiments were done. The 8-oxoG lesion is replicated in the second round; replication would be on the leading strand in R strains. Horizontal bars indicate selected genotype comparisons, with the letter above the bars indicating the probability of the null hypothesis that the results of transformation with Oligo GO or Oligo UGO in the two strains are the same. For a : P
    Figure Legend Snippet: Template switching induced by 8-oxoG. Template switching was determined as those transformants that did not contain the Sph I restriction site introduced by the oligo. More than 40 Trp+ revertants were assayed for the presence of an Sph I site in each strain of the indicated genotype. Error bars represent standard deviation of the mean for cases in which three or more independent experiments were done. The 8-oxoG lesion is replicated in the second round; replication would be on the leading strand in R strains. Horizontal bars indicate selected genotype comparisons, with the letter above the bars indicating the probability of the null hypothesis that the results of transformation with Oligo GO or Oligo UGO in the two strains are the same. For a : P

    Techniques Used: Standard Deviation, Transformation Assay

    Accuracy of 8-oxoG replication. Trp+ revertants containing an Sph I site were assayed for the presence of a Bfa I site resulting from insertion of an A opposite 8-oxoG in strains of the indicated genotype transformed with an 8-oxoG-containing oligo; those transformants lacking the Bfa I site had a C inserted opposite the 8-oxoG. In most cases, more than 40 Sph I containing revertants were assayed. Note that the 8-oxoG lesion is replicated in the second round; thus for example replication is on the leading strand in R strains. The error bars and horizontal bars are as in Figure 3 . For a : P
    Figure Legend Snippet: Accuracy of 8-oxoG replication. Trp+ revertants containing an Sph I site were assayed for the presence of a Bfa I site resulting from insertion of an A opposite 8-oxoG in strains of the indicated genotype transformed with an 8-oxoG-containing oligo; those transformants lacking the Bfa I site had a C inserted opposite the 8-oxoG. In most cases, more than 40 Sph I containing revertants were assayed. Note that the 8-oxoG lesion is replicated in the second round; thus for example replication is on the leading strand in R strains. The error bars and horizontal bars are as in Figure 3 . For a : P

    Techniques Used: Transformation Assay

    Sequences of TRP5 mutant regions and oligonucleotides used for reversion analysis. The trp5 - G148Cm mutant contains several changes designed to create additional completely degenerate third codon positions, with those of interest underlined; the sequence shown here begins at nt 128. The C at position 148 that must change to G in order to restore a Trp+ phenotype is highlighted in yellow. Oligo N creates 7 mismatches (highlighted in yellow and blue) upon annealing with the G148Cm sequence; oligos are numbered from the 5′ end. Oligo G and UG create a subset of those mismatches as indicated. Oligos GO and UGO are identical to Oligos G and UG except that the base highlighted in red is an 8-oxoG. If A is inserted opposite the 8-oxoG during replication, a novel Bfa I restriction site is created; similarly, the G highlighted in blue, if incorporated, creates a novel Sph I restriction site.
    Figure Legend Snippet: Sequences of TRP5 mutant regions and oligonucleotides used for reversion analysis. The trp5 - G148Cm mutant contains several changes designed to create additional completely degenerate third codon positions, with those of interest underlined; the sequence shown here begins at nt 128. The C at position 148 that must change to G in order to restore a Trp+ phenotype is highlighted in yellow. Oligo N creates 7 mismatches (highlighted in yellow and blue) upon annealing with the G148Cm sequence; oligos are numbered from the 5′ end. Oligo G and UG create a subset of those mismatches as indicated. Oligos GO and UGO are identical to Oligos G and UG except that the base highlighted in red is an 8-oxoG. If A is inserted opposite the 8-oxoG during replication, a novel Bfa I restriction site is created; similarly, the G highlighted in blue, if incorporated, creates a novel Sph I restriction site.

    Techniques Used: Mutagenesis, Sequencing

    4) Product Images from "The human deafness-associated connexin 30 T5M mutation causes mild hearing loss and reduces biochemical coupling among cochlear non-sensory cells in knock-in mice"

    Article Title: The human deafness-associated connexin 30 T5M mutation causes mild hearing loss and reduces biochemical coupling among cochlear non-sensory cells in knock-in mice

    Journal: Human Molecular Genetics

    doi: 10.1093/hmg/ddq402

    Generation of the Cx30T5M mice via homologous recombination. ( A ) Homologous recombination of the targeting vector into the genomic locus of Cx30 resulting in the mCx30T5M Neo locus. In mice, the neomycin resistance coding DNA is deleted by Flp recombinase activity resulting in the mCx30T5M locus. ( B ) PCR genotyping using the primers shown in (A). ( C ) PCR and subsequent digestion with Sph I to discriminate between wild-type and mutated Cx30 gene. ( D ) Southern blot hybridization indicating homologous recombination in mice.
    Figure Legend Snippet: Generation of the Cx30T5M mice via homologous recombination. ( A ) Homologous recombination of the targeting vector into the genomic locus of Cx30 resulting in the mCx30T5M Neo locus. In mice, the neomycin resistance coding DNA is deleted by Flp recombinase activity resulting in the mCx30T5M locus. ( B ) PCR genotyping using the primers shown in (A). ( C ) PCR and subsequent digestion with Sph I to discriminate between wild-type and mutated Cx30 gene. ( D ) Southern blot hybridization indicating homologous recombination in mice.

    Techniques Used: Mouse Assay, Homologous Recombination, Plasmid Preparation, Activity Assay, Polymerase Chain Reaction, Southern Blot, Hybridization

    5) Product Images from "DNA content analysis allows discrimination between Trypanosoma cruzi and Trypanosoma rangeli"

    Article Title: DNA content analysis allows discrimination between Trypanosoma cruzi and Trypanosoma rangeli

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0189907

    Genetic characterization Trypanosoma cruzi and Trypanosoma rangeli strains. (A) kDNA analysis of T . rangeli strains in a silver stained 6% polyacrylamide gel containing PCR products obtained with primers S35/S36/KP1L. The presence of a 165-bp band indicates the presence of KP1 minicircles. (B) Detection of the 832-bp fragment of TcSC5D gene in T . cruzi strains and clone. (C) PCR–RFLP of TcSC5D products digested with Hpa I and Sph I enzymes. MM: Molecular marker 100bp. NTC: No-template control.
    Figure Legend Snippet: Genetic characterization Trypanosoma cruzi and Trypanosoma rangeli strains. (A) kDNA analysis of T . rangeli strains in a silver stained 6% polyacrylamide gel containing PCR products obtained with primers S35/S36/KP1L. The presence of a 165-bp band indicates the presence of KP1 minicircles. (B) Detection of the 832-bp fragment of TcSC5D gene in T . cruzi strains and clone. (C) PCR–RFLP of TcSC5D products digested with Hpa I and Sph I enzymes. MM: Molecular marker 100bp. NTC: No-template control.

    Techniques Used: Staining, Polymerase Chain Reaction, Marker

    6) Product Images from "Improved Method for Rapid and Efficient Determination of Genome Replication and Protein Expression of Clinical Hepatitis B Virus Isolates ▿"

    Article Title: Improved Method for Rapid and Efficient Determination of Genome Replication and Protein Expression of Clinical Hepatitis B Virus Isolates ▿

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.02340-10

    Replication capacity of the HBV genome linearized by ApaI and SphI restriction enzymes. The EcoRI dimer of clone 4B was digested with ApaI or SphI, with or without further treatment with T4 DNA ligase before transfection into Huh7 cells. The uncut dimer
    Figure Legend Snippet: Replication capacity of the HBV genome linearized by ApaI and SphI restriction enzymes. The EcoRI dimer of clone 4B was digested with ApaI or SphI, with or without further treatment with T4 DNA ligase before transfection into Huh7 cells. The uncut dimer

    Techniques Used: Transfection

    7) Product Images from "Cleavage of a model DNA replication fork by a Type I restriction endonuclease"

    Article Title: Cleavage of a model DNA replication fork by a Type I restriction endonuclease

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp214

    Preparation of a long branched DNA (eF1). ( A ) pES1 was treated with SphI, PvuII and nicking endonuclease Nb.BbvCI. A partial fragment from pGap1 was prepared by PCR with indicated primers, followed by nicking with Nt.BbvCI. ( B ) Single strands between the nicks introduced in the previous step were dissociated by heating with removal by complementary oligo DNA from the fragment containing a gap structure. ( C ) Resulting DNAs with the gap were annealed to form a branched structure (Materials and methods section). A leftward triangle indicates an EcoR124I site, 5′ CGA TGCTGTA TTC . An open circle indicates 32 P for 5′-end labeling.
    Figure Legend Snippet: Preparation of a long branched DNA (eF1). ( A ) pES1 was treated with SphI, PvuII and nicking endonuclease Nb.BbvCI. A partial fragment from pGap1 was prepared by PCR with indicated primers, followed by nicking with Nt.BbvCI. ( B ) Single strands between the nicks introduced in the previous step were dissociated by heating with removal by complementary oligo DNA from the fragment containing a gap structure. ( C ) Resulting DNAs with the gap were annealed to form a branched structure (Materials and methods section). A leftward triangle indicates an EcoR124I site, 5′ CGA TGCTGTA TTC . An open circle indicates 32 P for 5′-end labeling.

    Techniques Used: Polymerase Chain Reaction, End Labeling

    8) Product Images from "Enhancement of UV Light Sensitivity of a Vibrio parahaemolyticus O3:K6 Pandemic Strain Due to Natural Lysogenization by a Telomeric Phage ▿"

    Article Title: Enhancement of UV Light Sensitivity of a Vibrio parahaemolyticus O3:K6 Pandemic Strain Due to Natural Lysogenization by a Telomeric Phage ▿

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.01995-08

    Restriction maps of plasmid and bacteriophage DNA obtained from V. parahaemolyticus strain PMC 58.5. N, H, and S correspond to NheI, HindIII, and SphI restriction enzymes sites, respectively. A hypothetical circular map is shown in the middle, with the possible ends shown with a red arrow and the restriction sites with a black line. b, bacteriophage; p, plasmid.
    Figure Legend Snippet: Restriction maps of plasmid and bacteriophage DNA obtained from V. parahaemolyticus strain PMC 58.5. N, H, and S correspond to NheI, HindIII, and SphI restriction enzymes sites, respectively. A hypothetical circular map is shown in the middle, with the possible ends shown with a red arrow and the restriction sites with a black line. b, bacteriophage; p, plasmid.

    Techniques Used: Plasmid Preparation

    9) Product Images from "Hygromycin B and Apramycin Antibiotic Resistance Cassettes for Use in Campylobacter jejuni"

    Article Title: Hygromycin B and Apramycin Antibiotic Resistance Cassettes for Use in Campylobacter jejuni

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0095084

    Synthesis of plasmids containing aph (7″) or aac (3)IV as non-polar hygromycin B and apramycin resistance markers. (A) Schematic of ultramers designed to amplify aph (7″) or aac (3)IV. The 5′ ultramers 5631 and 5633, for aph (7″) or aac (3)IV respectively, include Mfe I, Kpn I and Sma I restriction sites, stop codons in all three reading frames, and a ribosome binding site. The 3′ ultramers 5632 and 5634 include a ribosome binding site, a start codon in-frame with restriction sites for Sma I and Bam HI, and restriction sites for Xba I, Nde I, Pst I and Sph I. (B) The amplified aac (3)IV was introduced by TA cloning into linearized pGEM-T, conserving the restriction sites in the pGEM-T multiple cloning site (MCS). The resulting plasmid is pAC1A. The pGEM sites may also be used for the sub-cloning of the apramycin resistance marker ( Apr R ). MCS sites that cut aac (3)IV are indicated with a superscript ‘A’. (C) All introduced sites in pAC1A were tested by restriction digest. (D) The Mfe I- and Sph I-digested aph (7″) amplification product was cloned into pBAD24 digested with Eco RI ( Mfe I-compatible) and Sph I. The Mfe I site was lost in the resulting plasmid, pAC1H. (E) All restriction sites introduced to pAC1H were tested by digest.
    Figure Legend Snippet: Synthesis of plasmids containing aph (7″) or aac (3)IV as non-polar hygromycin B and apramycin resistance markers. (A) Schematic of ultramers designed to amplify aph (7″) or aac (3)IV. The 5′ ultramers 5631 and 5633, for aph (7″) or aac (3)IV respectively, include Mfe I, Kpn I and Sma I restriction sites, stop codons in all three reading frames, and a ribosome binding site. The 3′ ultramers 5632 and 5634 include a ribosome binding site, a start codon in-frame with restriction sites for Sma I and Bam HI, and restriction sites for Xba I, Nde I, Pst I and Sph I. (B) The amplified aac (3)IV was introduced by TA cloning into linearized pGEM-T, conserving the restriction sites in the pGEM-T multiple cloning site (MCS). The resulting plasmid is pAC1A. The pGEM sites may also be used for the sub-cloning of the apramycin resistance marker ( Apr R ). MCS sites that cut aac (3)IV are indicated with a superscript ‘A’. (C) All introduced sites in pAC1A were tested by restriction digest. (D) The Mfe I- and Sph I-digested aph (7″) amplification product was cloned into pBAD24 digested with Eco RI ( Mfe I-compatible) and Sph I. The Mfe I site was lost in the resulting plasmid, pAC1H. (E) All restriction sites introduced to pAC1H were tested by digest.

    Techniques Used: Binding Assay, Amplification, TA Cloning, Clone Assay, Plasmid Preparation, Subcloning, Marker

    10) Product Images from "A Simple Strain Typing Assay for Trypanosoma cruzi: Discrimination of Major Evolutionary Lineages from a Single Amplification Product"

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

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0001777

    Restriction fragment length polymorphism analysis of the TcMK amplification product. A: a multiple sequence alignment of the 5′-UTR just upstream of the translational start codon, showing the polymorphic Xho I site. A solid line box marks the Xho I site in TcV strains. B: Agarose gel electrophoresis showing a PCR-RFLP analysis of selected strains. Strains analyzed were: Sc43 cl9, MN cl2, LL014 and Teh53 for DTU TcV; and Tulahuen cl2, CL-Brener, Tul2 and P63 cl1 for DTU TcVI.
    Figure Legend Snippet: Restriction fragment length polymorphism analysis of the TcMK amplification product. A: a multiple sequence alignment of the 5′-UTR just upstream of the translational start codon, showing the polymorphic Xho I site. A solid line box marks the Xho I site in TcV strains. B: Agarose gel electrophoresis showing a PCR-RFLP analysis of selected strains. Strains analyzed were: Sc43 cl9, MN cl2, LL014 and Teh53 for DTU TcV; and Tulahuen cl2, CL-Brener, Tul2 and P63 cl1 for DTU TcVI.

    Techniques Used: Amplification, Sequencing, Agarose Gel Electrophoresis, Polymerase Chain Reaction

    11) Product Images from "DNA content analysis allows discrimination between Trypanosoma cruzi and Trypanosoma rangeli"

    Article Title: DNA content analysis allows discrimination between Trypanosoma cruzi and Trypanosoma rangeli

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0189907

    Genetic characterization Trypanosoma cruzi and Trypanosoma rangeli strains. (A) kDNA analysis of T . rangeli strains in a silver stained 6% polyacrylamide gel containing PCR products obtained with primers S35/S36/KP1L. The presence of a 165-bp band indicates the presence of KP1 minicircles. (B) Detection of the 832-bp fragment of TcSC5D gene in T . cruzi strains and clone. (C) PCR–RFLP of TcSC5D products digested with Hpa I and Sph I enzymes. MM: Molecular marker 100bp. NTC: No-template control.
    Figure Legend Snippet: Genetic characterization Trypanosoma cruzi and Trypanosoma rangeli strains. (A) kDNA analysis of T . rangeli strains in a silver stained 6% polyacrylamide gel containing PCR products obtained with primers S35/S36/KP1L. The presence of a 165-bp band indicates the presence of KP1 minicircles. (B) Detection of the 832-bp fragment of TcSC5D gene in T . cruzi strains and clone. (C) PCR–RFLP of TcSC5D products digested with Hpa I and Sph I enzymes. MM: Molecular marker 100bp. NTC: No-template control.

    Techniques Used: Staining, Polymerase Chain Reaction, Marker

    12) Product Images from "DNA content analysis allows discrimination between Trypanosoma cruzi and Trypanosoma rangeli"

    Article Title: DNA content analysis allows discrimination between Trypanosoma cruzi and Trypanosoma rangeli

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0189907

    Genetic characterization Trypanosoma cruzi and Trypanosoma rangeli strains. (A) kDNA analysis of T . rangeli strains in a silver stained 6% polyacrylamide gel containing PCR products obtained with primers S35/S36/KP1L. The presence of a 165-bp band indicates the presence of KP1 minicircles. (B) Detection of the 832-bp fragment of TcSC5D gene in T . cruzi strains and clone. (C) PCR–RFLP of TcSC5D products digested with Hpa I and Sph I enzymes. MM: Molecular marker 100bp. NTC: No-template control.
    Figure Legend Snippet: Genetic characterization Trypanosoma cruzi and Trypanosoma rangeli strains. (A) kDNA analysis of T . rangeli strains in a silver stained 6% polyacrylamide gel containing PCR products obtained with primers S35/S36/KP1L. The presence of a 165-bp band indicates the presence of KP1 minicircles. (B) Detection of the 832-bp fragment of TcSC5D gene in T . cruzi strains and clone. (C) PCR–RFLP of TcSC5D products digested with Hpa I and Sph I enzymes. MM: Molecular marker 100bp. NTC: No-template control.

    Techniques Used: Staining, Polymerase Chain Reaction, Marker

    13) Product Images from "Two single nucleotide polymorphisms sites in α1-AT gene and their association with somatic cell score in Chinese Holstein cows"

    Article Title: Two single nucleotide polymorphisms sites in α1-AT gene and their association with somatic cell score in Chinese Holstein cows

    Journal: Journal of Biological Research

    doi: 10.1186/s40709-017-0065-z

    Sph I-RFLP patterns of PCR products of G5503A site in 2% agarose gel. Note 6, 8, 9, 12: AA genotype; 1, 4, 7, 10, 11: AB genotype; 2, 3, 5, 13; BB genotype; M: D2000 DNA marker
    Figure Legend Snippet: Sph I-RFLP patterns of PCR products of G5503A site in 2% agarose gel. Note 6, 8, 9, 12: AA genotype; 1, 4, 7, 10, 11: AB genotype; 2, 3, 5, 13; BB genotype; M: D2000 DNA marker

    Techniques Used: Polymerase Chain Reaction, Agarose Gel Electrophoresis, Marker

    14) Product Images from "Gelatin nanoparticles enhance delivery of hepatitis C virus recombinant NS2 gene"

    Article Title: Gelatin nanoparticles enhance delivery of hepatitis C virus recombinant NS2 gene

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0181723

    Agarose gel electrophoresis for miniprep samples of the recombinant PQE 30/ NS2 plasmid/Gel.NPs double digested ( SphI / HindIII ) showing two bands; band for NS2 insert (650 bp) and band for pQE-30 vector (3.4 kb) in Lane 1 and 2. Lane M: 1 kb DNA marker.
    Figure Legend Snippet: Agarose gel electrophoresis for miniprep samples of the recombinant PQE 30/ NS2 plasmid/Gel.NPs double digested ( SphI / HindIII ) showing two bands; band for NS2 insert (650 bp) and band for pQE-30 vector (3.4 kb) in Lane 1 and 2. Lane M: 1 kb DNA marker.

    Techniques Used: Agarose Gel Electrophoresis, Recombinant, Plasmid Preparation, Marker

    Related Articles

    Clone Assay:

    Article Title: Improved Method for Rapid and Efficient Determination of Genome Replication and Protein Expression of Clinical Hepatitis B Virus Isolates ▿
    Article Snippet: .. A single copy of the full-length HBV genome was released by the EcoRI digestion of the EcoRI monomer, ApaI or SphI digestion of the EcoRI dimer, and digestion of the monomeric PCR clones or clone pools at 50°C with BspQI (New England BioLabs). .. The digested DNA was purified through QIAquick PCR purification columns (Qiagen) and resuspended in endotoxin-free Tris-EDTA buffer.

    Amplification:

    Article Title: DNA content analysis allows discrimination between Trypanosoma cruzi and Trypanosoma rangeli
    Article Snippet: .. Aliquots of 20 μL of the amplified products were digested with 1U of the enzyme Hpa I (NEB R105) at 55°C for 1 h and with 1U of the enzyme Sph I (NEB R0182) at 37°C for 1 h (TcSC5D fragment) or with one unit of Xho I (NEB R0146S) endonuclease (TcMK fragment). .. The resulting digestion fragments were observed on 1.5% agarose gel and stained with ethidium bromide.

    Article Title: The human deafness-associated connexin 30 T5M mutation causes mild hearing loss and reduces biochemical coupling among cochlear non-sensory cells in knock-in mice
    Article Snippet: .. After completing the PCR 5 μl of ‘buffer 2’ and 1 μl of Sph I (New England Biolabs, Ipswich, USA) were added and the complete mix was incubated for at 37°C 3 h. The PCR product was demonstrated as a 1436 bp amplicon, and the presence of the mutation was proven by four additional bands at 1184, 594, 590 and 252 bp. .. The genotypes were further characterized by Southern blot hybridization.

    Nucleic Acid Electrophoresis:

    Article Title: In Vivo Bypass of 8-oxodG
    Article Snippet: .. For restriction digestion, 5 µl of the PCR reaction was incubated with 2 units of either Bfa I or Sph I (New England Biolabs) in the recommended buffer in a total volume of 15 µl at 37° overnight and analyzed by gel electrophoresis. ..

    Mutagenesis:

    Article Title: The human deafness-associated connexin 30 T5M mutation causes mild hearing loss and reduces biochemical coupling among cochlear non-sensory cells in knock-in mice
    Article Snippet: .. After completing the PCR 5 μl of ‘buffer 2’ and 1 μl of Sph I (New England Biolabs, Ipswich, USA) were added and the complete mix was incubated for at 37°C 3 h. The PCR product was demonstrated as a 1436 bp amplicon, and the presence of the mutation was proven by four additional bands at 1184, 594, 590 and 252 bp. .. The genotypes were further characterized by Southern blot hybridization.

    Purification:

    Article Title: Hygromycin B and Apramycin Antibiotic Resistance Cassettes for Use in Campylobacter jejuni
    Article Snippet: .. The initial aph (7″) PCR product was instead digested with Mfe I and Sph I (NEB), purified, and ligated to low-copy pBAD24 digested with Eco RI and Sph I. .. The ligation was transformed into E. coli DH5α and sequencing of the transformants indicated the correct aph (7″) sequence was incorporated.

    Incubation:

    Article Title: The human deafness-associated connexin 30 T5M mutation causes mild hearing loss and reduces biochemical coupling among cochlear non-sensory cells in knock-in mice
    Article Snippet: .. After completing the PCR 5 μl of ‘buffer 2’ and 1 μl of Sph I (New England Biolabs, Ipswich, USA) were added and the complete mix was incubated for at 37°C 3 h. The PCR product was demonstrated as a 1436 bp amplicon, and the presence of the mutation was proven by four additional bands at 1184, 594, 590 and 252 bp. .. The genotypes were further characterized by Southern blot hybridization.

    Article Title: In Vivo Bypass of 8-oxodG
    Article Snippet: .. For restriction digestion, 5 µl of the PCR reaction was incubated with 2 units of either Bfa I or Sph I (New England Biolabs) in the recommended buffer in a total volume of 15 µl at 37° overnight and analyzed by gel electrophoresis. ..

    Polymerase Chain Reaction:

    Article Title: The human deafness-associated connexin 30 T5M mutation causes mild hearing loss and reduces biochemical coupling among cochlear non-sensory cells in knock-in mice
    Article Snippet: .. After completing the PCR 5 μl of ‘buffer 2’ and 1 μl of Sph I (New England Biolabs, Ipswich, USA) were added and the complete mix was incubated for at 37°C 3 h. The PCR product was demonstrated as a 1436 bp amplicon, and the presence of the mutation was proven by four additional bands at 1184, 594, 590 and 252 bp. .. The genotypes were further characterized by Southern blot hybridization.

    Article Title: Improved Method for Rapid and Efficient Determination of Genome Replication and Protein Expression of Clinical Hepatitis B Virus Isolates ▿
    Article Snippet: .. A single copy of the full-length HBV genome was released by the EcoRI digestion of the EcoRI monomer, ApaI or SphI digestion of the EcoRI dimer, and digestion of the monomeric PCR clones or clone pools at 50°C with BspQI (New England BioLabs). .. The digested DNA was purified through QIAquick PCR purification columns (Qiagen) and resuspended in endotoxin-free Tris-EDTA buffer.

    Article Title: Hygromycin B and Apramycin Antibiotic Resistance Cassettes for Use in Campylobacter jejuni
    Article Snippet: .. The initial aph (7″) PCR product was instead digested with Mfe I and Sph I (NEB), purified, and ligated to low-copy pBAD24 digested with Eco RI and Sph I. .. The ligation was transformed into E. coli DH5α and sequencing of the transformants indicated the correct aph (7″) sequence was incorporated.

    Article Title: In Vivo Bypass of 8-oxodG
    Article Snippet: .. For restriction digestion, 5 µl of the PCR reaction was incubated with 2 units of either Bfa I or Sph I (New England Biolabs) in the recommended buffer in a total volume of 15 µl at 37° overnight and analyzed by gel electrophoresis. ..

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    New England Biolabs sphi
    Flow chart for the generation of replication-competent HBV genomes from blood samples. Virion-associated HBV DNA has the minus strand DNA ( thick line ) complete. The sense (S) primer anneals to its 3’ end to generate full-length plus strand, which will serve as the template for the antisense (AS) primer to generate more minus strand DNA. The HindIII and SacI sites introduced to the sense and antisense primers, respectively, will allow efficient cloning of the PCR product to pUC18 vector, whereas the internal <t>BspQI</t> sites allow subsequent precise release of the HBV genome. Such a linear HBV genome can be ligated in vitro to make it replication competent (capable of producing the terminally redundant pg RNA), or the ligated DNA is further digested with <t>SphI</t> and ligated with SphI cut, dephosphorylated pUC18 DNA. Bacterial colonies harboring tandem SphI dimer can be screened by hybridization with an oligoprobe spanning the SphI site. The 3.5-kb pg RNA can be produced from such a tandem dimer construct
    Sphi, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 89 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Flow chart for the generation of replication-competent HBV genomes from blood samples. Virion-associated HBV DNA has the minus strand DNA ( thick line ) complete. The sense (S) primer anneals to its 3’ end to generate full-length plus strand, which will serve as the template for the antisense (AS) primer to generate more minus strand DNA. The HindIII and SacI sites introduced to the sense and antisense primers, respectively, will allow efficient cloning of the PCR product to pUC18 vector, whereas the internal BspQI sites allow subsequent precise release of the HBV genome. Such a linear HBV genome can be ligated in vitro to make it replication competent (capable of producing the terminally redundant pg RNA), or the ligated DNA is further digested with SphI and ligated with SphI cut, dephosphorylated pUC18 DNA. Bacterial colonies harboring tandem SphI dimer can be screened by hybridization with an oligoprobe spanning the SphI site. The 3.5-kb pg RNA can be produced from such a tandem dimer construct

    Journal: Methods in molecular biology (Clifton, N.J.)

    Article Title: Generation of Replication-Competent Hepatitis B Virus Genome from Blood Samples for Functional Characterization

    doi: 10.1007/978-1-4939-6700-1_18

    Figure Lengend Snippet: Flow chart for the generation of replication-competent HBV genomes from blood samples. Virion-associated HBV DNA has the minus strand DNA ( thick line ) complete. The sense (S) primer anneals to its 3’ end to generate full-length plus strand, which will serve as the template for the antisense (AS) primer to generate more minus strand DNA. The HindIII and SacI sites introduced to the sense and antisense primers, respectively, will allow efficient cloning of the PCR product to pUC18 vector, whereas the internal BspQI sites allow subsequent precise release of the HBV genome. Such a linear HBV genome can be ligated in vitro to make it replication competent (capable of producing the terminally redundant pg RNA), or the ligated DNA is further digested with SphI and ligated with SphI cut, dephosphorylated pUC18 DNA. Bacterial colonies harboring tandem SphI dimer can be screened by hybridization with an oligoprobe spanning the SphI site. The 3.5-kb pg RNA can be produced from such a tandem dimer construct

    Article Snippet: Enzymes: BspQI, HindIII, SacI, ScaI, SphI, alkaline phosphatase, and Q5 DNA polymerase (New England Biolabs).

    Techniques: Flow Cytometry, Clone Assay, Polymerase Chain Reaction, Plasmid Preparation, In Vitro, Hybridization, Produced, Construct

    Models for incorporation of oligo GO and UGO. (A) Incorporation of Oligo GO creates three mismatches; if the oligo is not removed before the second round, replication of the C, indicated in yellow, would result in a Trp+ phenotype, replication of the 8-oxoG, indicated in red, would create a Bfa I site if an A were incorporated, and replication of the G indicated in blue would create an Sph I site. In the first round, usually the entire portion of the oligo containing the marked bases remains or is removed by MMR. However, when MMR is present, the segment of the oligo containing the 5′-two mismatches can be removed, but the segment creating the Trp+ phenotype can be left, presumably due to failure of MMR to recognized the C-C mismatch (see text). If the marked segment of Oligo GO persists to the second round of replication, translesion synthesis creates an Sph I site and a Bfa I site if the 8-oxoG is bypassed with an A. If the 8-oxoG induces template switching, no Sph I site is created. (B) Incorporation of Oligo UGO creates an additional mismatch with the C indicated in blue, 3′ of the C-C mismatch. Due to this additional mismatch, in the first round, either the entire oligo remains, or is removed. The results of the second round of replication are identical to that of Oligo GO in (A). Cells with a gray background are Trp- and thus do not survive selection.

    Journal: PLoS Genetics

    Article Title: In Vivo Bypass of 8-oxodG

    doi: 10.1371/journal.pgen.1003682

    Figure Lengend Snippet: Models for incorporation of oligo GO and UGO. (A) Incorporation of Oligo GO creates three mismatches; if the oligo is not removed before the second round, replication of the C, indicated in yellow, would result in a Trp+ phenotype, replication of the 8-oxoG, indicated in red, would create a Bfa I site if an A were incorporated, and replication of the G indicated in blue would create an Sph I site. In the first round, usually the entire portion of the oligo containing the marked bases remains or is removed by MMR. However, when MMR is present, the segment of the oligo containing the 5′-two mismatches can be removed, but the segment creating the Trp+ phenotype can be left, presumably due to failure of MMR to recognized the C-C mismatch (see text). If the marked segment of Oligo GO persists to the second round of replication, translesion synthesis creates an Sph I site and a Bfa I site if the 8-oxoG is bypassed with an A. If the 8-oxoG induces template switching, no Sph I site is created. (B) Incorporation of Oligo UGO creates an additional mismatch with the C indicated in blue, 3′ of the C-C mismatch. Due to this additional mismatch, in the first round, either the entire oligo remains, or is removed. The results of the second round of replication are identical to that of Oligo GO in (A). Cells with a gray background are Trp- and thus do not survive selection.

    Article Snippet: For restriction digestion, 5 µl of the PCR reaction was incubated with 2 units of either Bfa I or Sph I (New England Biolabs) in the recommended buffer in a total volume of 15 µl at 37° overnight and analyzed by gel electrophoresis.

    Techniques: Translesion Synthesis, Selection

    Template switching induced by 8-oxoG. Template switching was determined as those transformants that did not contain the Sph I restriction site introduced by the oligo. More than 40 Trp+ revertants were assayed for the presence of an Sph I site in each strain of the indicated genotype. Error bars represent standard deviation of the mean for cases in which three or more independent experiments were done. The 8-oxoG lesion is replicated in the second round; replication would be on the leading strand in R strains. Horizontal bars indicate selected genotype comparisons, with the letter above the bars indicating the probability of the null hypothesis that the results of transformation with Oligo GO or Oligo UGO in the two strains are the same. For a : P

    Journal: PLoS Genetics

    Article Title: In Vivo Bypass of 8-oxodG

    doi: 10.1371/journal.pgen.1003682

    Figure Lengend Snippet: Template switching induced by 8-oxoG. Template switching was determined as those transformants that did not contain the Sph I restriction site introduced by the oligo. More than 40 Trp+ revertants were assayed for the presence of an Sph I site in each strain of the indicated genotype. Error bars represent standard deviation of the mean for cases in which three or more independent experiments were done. The 8-oxoG lesion is replicated in the second round; replication would be on the leading strand in R strains. Horizontal bars indicate selected genotype comparisons, with the letter above the bars indicating the probability of the null hypothesis that the results of transformation with Oligo GO or Oligo UGO in the two strains are the same. For a : P

    Article Snippet: For restriction digestion, 5 µl of the PCR reaction was incubated with 2 units of either Bfa I or Sph I (New England Biolabs) in the recommended buffer in a total volume of 15 µl at 37° overnight and analyzed by gel electrophoresis.

    Techniques: Standard Deviation, Transformation Assay

    Accuracy of 8-oxoG replication. Trp+ revertants containing an Sph I site were assayed for the presence of a Bfa I site resulting from insertion of an A opposite 8-oxoG in strains of the indicated genotype transformed with an 8-oxoG-containing oligo; those transformants lacking the Bfa I site had a C inserted opposite the 8-oxoG. In most cases, more than 40 Sph I containing revertants were assayed. Note that the 8-oxoG lesion is replicated in the second round; thus for example replication is on the leading strand in R strains. The error bars and horizontal bars are as in Figure 3 . For a : P

    Journal: PLoS Genetics

    Article Title: In Vivo Bypass of 8-oxodG

    doi: 10.1371/journal.pgen.1003682

    Figure Lengend Snippet: Accuracy of 8-oxoG replication. Trp+ revertants containing an Sph I site were assayed for the presence of a Bfa I site resulting from insertion of an A opposite 8-oxoG in strains of the indicated genotype transformed with an 8-oxoG-containing oligo; those transformants lacking the Bfa I site had a C inserted opposite the 8-oxoG. In most cases, more than 40 Sph I containing revertants were assayed. Note that the 8-oxoG lesion is replicated in the second round; thus for example replication is on the leading strand in R strains. The error bars and horizontal bars are as in Figure 3 . For a : P

    Article Snippet: For restriction digestion, 5 µl of the PCR reaction was incubated with 2 units of either Bfa I or Sph I (New England Biolabs) in the recommended buffer in a total volume of 15 µl at 37° overnight and analyzed by gel electrophoresis.

    Techniques: Transformation Assay

    Sequences of TRP5 mutant regions and oligonucleotides used for reversion analysis. The trp5 - G148Cm mutant contains several changes designed to create additional completely degenerate third codon positions, with those of interest underlined; the sequence shown here begins at nt 128. The C at position 148 that must change to G in order to restore a Trp+ phenotype is highlighted in yellow. Oligo N creates 7 mismatches (highlighted in yellow and blue) upon annealing with the G148Cm sequence; oligos are numbered from the 5′ end. Oligo G and UG create a subset of those mismatches as indicated. Oligos GO and UGO are identical to Oligos G and UG except that the base highlighted in red is an 8-oxoG. If A is inserted opposite the 8-oxoG during replication, a novel Bfa I restriction site is created; similarly, the G highlighted in blue, if incorporated, creates a novel Sph I restriction site.

    Journal: PLoS Genetics

    Article Title: In Vivo Bypass of 8-oxodG

    doi: 10.1371/journal.pgen.1003682

    Figure Lengend Snippet: Sequences of TRP5 mutant regions and oligonucleotides used for reversion analysis. The trp5 - G148Cm mutant contains several changes designed to create additional completely degenerate third codon positions, with those of interest underlined; the sequence shown here begins at nt 128. The C at position 148 that must change to G in order to restore a Trp+ phenotype is highlighted in yellow. Oligo N creates 7 mismatches (highlighted in yellow and blue) upon annealing with the G148Cm sequence; oligos are numbered from the 5′ end. Oligo G and UG create a subset of those mismatches as indicated. Oligos GO and UGO are identical to Oligos G and UG except that the base highlighted in red is an 8-oxoG. If A is inserted opposite the 8-oxoG during replication, a novel Bfa I restriction site is created; similarly, the G highlighted in blue, if incorporated, creates a novel Sph I restriction site.

    Article Snippet: For restriction digestion, 5 µl of the PCR reaction was incubated with 2 units of either Bfa I or Sph I (New England Biolabs) in the recommended buffer in a total volume of 15 µl at 37° overnight and analyzed by gel electrophoresis.

    Techniques: Mutagenesis, Sequencing

    Generation of the Cx30T5M mice via homologous recombination. ( A ) Homologous recombination of the targeting vector into the genomic locus of Cx30 resulting in the mCx30T5M Neo locus. In mice, the neomycin resistance coding DNA is deleted by Flp recombinase activity resulting in the mCx30T5M locus. ( B ) PCR genotyping using the primers shown in (A). ( C ) PCR and subsequent digestion with Sph I to discriminate between wild-type and mutated Cx30 gene. ( D ) Southern blot hybridization indicating homologous recombination in mice.

    Journal: Human Molecular Genetics

    Article Title: The human deafness-associated connexin 30 T5M mutation causes mild hearing loss and reduces biochemical coupling among cochlear non-sensory cells in knock-in mice

    doi: 10.1093/hmg/ddq402

    Figure Lengend Snippet: Generation of the Cx30T5M mice via homologous recombination. ( A ) Homologous recombination of the targeting vector into the genomic locus of Cx30 resulting in the mCx30T5M Neo locus. In mice, the neomycin resistance coding DNA is deleted by Flp recombinase activity resulting in the mCx30T5M locus. ( B ) PCR genotyping using the primers shown in (A). ( C ) PCR and subsequent digestion with Sph I to discriminate between wild-type and mutated Cx30 gene. ( D ) Southern blot hybridization indicating homologous recombination in mice.

    Article Snippet: After completing the PCR 5 μl of ‘buffer 2’ and 1 μl of Sph I (New England Biolabs, Ipswich, USA) were added and the complete mix was incubated for at 37°C 3 h. The PCR product was demonstrated as a 1436 bp amplicon, and the presence of the mutation was proven by four additional bands at 1184, 594, 590 and 252 bp.

    Techniques: Mouse Assay, Homologous Recombination, Plasmid Preparation, Activity Assay, Polymerase Chain Reaction, Southern Blot, Hybridization

    Replication capacity of the HBV genome linearized by ApaI and SphI restriction enzymes. The EcoRI dimer of clone 4B was digested with ApaI or SphI, with or without further treatment with T4 DNA ligase before transfection into Huh7 cells. The uncut dimer

    Journal: Journal of Clinical Microbiology

    Article Title: Improved Method for Rapid and Efficient Determination of Genome Replication and Protein Expression of Clinical Hepatitis B Virus Isolates ▿

    doi: 10.1128/JCM.02340-10

    Figure Lengend Snippet: Replication capacity of the HBV genome linearized by ApaI and SphI restriction enzymes. The EcoRI dimer of clone 4B was digested with ApaI or SphI, with or without further treatment with T4 DNA ligase before transfection into Huh7 cells. The uncut dimer

    Article Snippet: A single copy of the full-length HBV genome was released by the EcoRI digestion of the EcoRI monomer, ApaI or SphI digestion of the EcoRI dimer, and digestion of the monomeric PCR clones or clone pools at 50°C with BspQI (New England BioLabs).

    Techniques: Transfection