sphi  (New England Biolabs)


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    Name:
    SphI
    Description:
    SphI 2 500 units
    Catalog Number:
    R0182L
    Price:
    282
    Category:
    Restriction Enzymes
    Size:
    2 500 units
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    Structured Review

    New England Biolabs sphi
    SphI
    SphI 2 500 units
    https://www.bioz.com/result/sphi/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    sphi - by Bioz Stars, 2021-04
    99/100 stars

    Images

    1) 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

    2) 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

    3) 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

    4) 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

    Related Articles

    Polymerase Chain Reaction:

    Article Title: In Vivo Bypass of 8-oxodG
    Article Snippet: PCR was performed using 5 µl of the lysate in a total volume of 50 µl of the recommended buffer with 0.3 µM trpseq2 and trpseq8 primers and 0.5 µl Takara e2TAK DNA polymerase for 30 cycles. .. 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. ..

    Article Title: Two single nucleotide polymorphisms sites in α1-AT gene and their association with somatic cell score in Chinese Holstein cows
    Article Snippet: Based on the results of sequence of P1, one mutation was revealed in exon 3 and restriction enzyme Sph I was used to detect it. .. The PCR products of P3 were digested by Sph I (NEB, Beijing, China) with 10 μl volume containing 5 μl of PCR product, 0.5 μl of 10 U µl−1 restriction enzyme Sph I, 1 μl of ×10 reaction buffer, 3.5 μl H2 O, incubated at 37 °C for 5 h. The mixtures were detected by 3.0% agarose gels and were genotyped. .. Creating restriction site (CRS) When there was no available restriction enzyme site for PCR-RFLP analysis, CRS combined with PCR amplification is a simple and efficient method that could be used to detect single nucleotide polymorphisms (SNPs) genotypes [ ].

    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: The T5M mutation was detected by PCR analysis using the primers Cx30_for (5′-GTC AAT TAA TGG CAT TGT TTC ACC-3′ and Cx30_rev (5′-CGG GAT CCA TGC ATC AGA TCA ATG TTG TCT ACA AAG AGG-3′) followed by a digestion with the restriction enzyme Sph I. PCR conditions were: 5 min at 94°C, 35 cycles of 30 c at 94°C, 1 min at 60°C, 1 min at 72°C, followed by 10 min at 72°C. .. 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: Hygromycin B and Apramycin Antibiotic Resistance Cassettes for Use in Campylobacter jejuni
    Article Snippet: Sequencing of pGEM-T containing aph (7″) indicated that the restriction sites flanking aph (7″) and aph (7″) sequence itself were incorrect. .. 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: In Vivo Bypass of 8-oxodG
    Article Snippet: PCR was performed using 5 µl of the lysate in a total volume of 50 µl of the recommended buffer with 0.3 µM trpseq2 and trpseq8 primers and 0.5 µl Takara e2TAK DNA polymerase for 30 cycles. .. 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. ..

    Article Title: Two single nucleotide polymorphisms sites in α1-AT gene and their association with somatic cell score in Chinese Holstein cows
    Article Snippet: Based on the results of sequence of P1, one mutation was revealed in exon 3 and restriction enzyme Sph I was used to detect it. .. The PCR products of P3 were digested by Sph I (NEB, Beijing, China) with 10 μl volume containing 5 μl of PCR product, 0.5 μl of 10 U µl−1 restriction enzyme Sph I, 1 μl of ×10 reaction buffer, 3.5 μl H2 O, incubated at 37 °C for 5 h. The mixtures were detected by 3.0% agarose gels and were genotyped. .. Creating restriction site (CRS) When there was no available restriction enzyme site for PCR-RFLP analysis, CRS combined with PCR amplification is a simple and efficient method that could be used to detect single nucleotide polymorphisms (SNPs) genotypes [ ].

    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: The T5M mutation was detected by PCR analysis using the primers Cx30_for (5′-GTC AAT TAA TGG CAT TGT TTC ACC-3′ and Cx30_rev (5′-CGG GAT CCA TGC ATC AGA TCA ATG TTG TCT ACA AAG AGG-3′) followed by a digestion with the restriction enzyme Sph I. PCR conditions were: 5 min at 94°C, 35 cycles of 30 c at 94°C, 1 min at 60°C, 1 min at 72°C, followed by 10 min at 72°C. .. 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: PCR was performed using 5 µl of the lysate in a total volume of 50 µl of the recommended buffer with 0.3 µM trpseq2 and trpseq8 primers and 0.5 µl Takara e2TAK DNA polymerase for 30 cycles. .. 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. ..

    Amplification:

    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: The T5M mutation was detected by PCR analysis using the primers Cx30_for (5′-GTC AAT TAA TGG CAT TGT TTC ACC-3′ and Cx30_rev (5′-CGG GAT CCA TGC ATC AGA TCA ATG TTG TCT ACA AAG AGG-3′) followed by a digestion with the restriction enzyme Sph I. PCR conditions were: 5 min at 94°C, 35 cycles of 30 c at 94°C, 1 min at 60°C, 1 min at 72°C, followed by 10 min at 72°C. .. 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.

    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: The T5M mutation was detected by PCR analysis using the primers Cx30_for (5′-GTC AAT TAA TGG CAT TGT TTC ACC-3′ and Cx30_rev (5′-CGG GAT CCA TGC ATC AGA TCA ATG TTG TCT ACA AAG AGG-3′) followed by a digestion with the restriction enzyme Sph I. PCR conditions were: 5 min at 94°C, 35 cycles of 30 c at 94°C, 1 min at 60°C, 1 min at 72°C, followed by 10 min at 72°C. .. 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: Sequencing of pGEM-T containing aph (7″) indicated that the restriction sites flanking aph (7″) and aph (7″) sequence itself were incorrect. .. 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.

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    New England Biolabs sph i
    Sph I-RFLP patterns of <t>PCR</t> 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
    Sph I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sph i/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    sph i - by Bioz Stars, 2021-04
    99/100 stars
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    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

    Journal: Journal of Biological Research

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

    doi: 10.1186/s40709-017-0065-z

    Figure Lengend 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

    Article Snippet: The PCR products of P3 were digested by Sph I (NEB, Beijing, China) with 10 μl volume containing 5 μl of PCR product, 0.5 μl of 10 U µl−1 restriction enzyme Sph I, 1 μl of ×10 reaction buffer, 3.5 μl H2 O, incubated at 37 °C for 5 h. The mixtures were detected by 3.0% agarose gels and were genotyped.

    Techniques: Polymerase Chain Reaction, Agarose Gel Electrophoresis, Marker

    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

    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.

    Journal: Applied and Environmental Microbiology

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

    doi: 10.1128/AEM.01995-08

    Figure Lengend 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.

    Article Snippet: Restriction site mapping was performed by digestion with restriction enzymes NheI, HindIII, and SphI from New England Biolabs (Massachusetts), according to the manufacturer's instructions.

    Techniques: Plasmid Preparation

    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.

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0095084

    Figure Lengend 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.

    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.

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