sbfi  (New England Biolabs)


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    Name:
    SbfI
    Description:
    SbfI 2 500 units
    Catalog Number:
    r0642l
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    302
    Size:
    2 500 units
    Category:
    Restriction Enzymes
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    Structured Review

    New England Biolabs sbfi
    SbfI
    SbfI 2 500 units
    https://www.bioz.com/result/sbfi/product/New England Biolabs
    Average 99 stars, based on 24 article reviews
    Price from $9.99 to $1999.99
    sbfi - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "Conserved CxnC Motifs in Kaposi’s Sarcoma-Associated Herpesvirus ORF66 Are Required for Viral Late Gene Expression and Are Essential for Its Interaction with ORF34"

    Article Title: Conserved CxnC Motifs in Kaposi’s Sarcoma-Associated Herpesvirus ORF66 Are Required for Viral Late Gene Expression and Are Essential for Its Interaction with ORF34

    Journal: Journal of Virology

    doi: 10.1128/JVI.01299-19

    ORF66 is essential in KSHV and required for late gene transcription. (A) (Left) Diagram showing the genomic locus of ORF66 with surrounding genes ORF67 (which partially overlaps ORF66) and ORF65, depicting the location of introduced mutations. (Right) The mutations were confirmed by Sanger sequencing. (B) Digestion of the recombinant BACs with SbfI or RsrII demonstrates that the introduction of mutations did not introduce large-scale changes. (C) Infectious virion production was measured by supernatant transfer from reactivated iSLK cell lines followed by flow cytometry. Data are from three independent biological replicates, with statistics being calculated using an unpaired t test. ****, P
    Figure Legend Snippet: ORF66 is essential in KSHV and required for late gene transcription. (A) (Left) Diagram showing the genomic locus of ORF66 with surrounding genes ORF67 (which partially overlaps ORF66) and ORF65, depicting the location of introduced mutations. (Right) The mutations were confirmed by Sanger sequencing. (B) Digestion of the recombinant BACs with SbfI or RsrII demonstrates that the introduction of mutations did not introduce large-scale changes. (C) Infectious virion production was measured by supernatant transfer from reactivated iSLK cell lines followed by flow cytometry. Data are from three independent biological replicates, with statistics being calculated using an unpaired t test. ****, P

    Techniques Used: Sequencing, Recombinant, Introduce, Flow Cytometry

    2) Product Images from "Secretoglobin 1A1 and 1A1A Differentially Regulate Neutrophil Reactive Oxygen Species Production, Phagocytosis and Extracellular Trap Formation"

    Article Title: Secretoglobin 1A1 and 1A1A Differentially Regulate Neutrophil Reactive Oxygen Species Production, Phagocytosis and Extracellular Trap Formation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0096217

    Cloning, expression and purification of equine recombinant SCGB 1A1 and SCGB 1A1A proteins. (A) SCGB1A1 (“1”) and SCGB1A1A (“1A”) partial ORFs were amplified from equine lung cDNA preparations. A unique band of appropriate size (225 bp) was amplified for each gene. L = 1 Kb+ DNA ladder. (B) Fragments were digested with XmnI and SbfI restriction enzymes (purple boxes) and inserted into the multiple cloning sites (MCS) of the pMAL-c5X expression vector (top). DNA from the transformed colonies was submitted for sequencing to determine the presence, integrity, orientation and suitable translational reading frame of the insert. SCGB1A1 and SCGB1A1A sequenced (S) products showed proper orientation and 100% identity to the predicted (P) sequences. (C) Fractions collected during the purification steps of SCGB 1A1 and SCGB 1A1A were analyzed by SDS-PAGE. A fusion protein was apparent in extracts from IPTG-induced (I) but not un-induced (U) colonies. A crude extract (CE) was collected from induced cells and purified by affinity chromatography, using an amylose (A) column. The eluted fractions were pooled and incubated with Factor Xa protease (Fx) to cleave the fusion proteins. Fx was removed by FPLC (F), and MBP (42.5 kDa) was removed by additional passage on an amylose column from which pure (P) recombinant proteins (7 kDa) were collected. (D) Purified SCGB 1A1 and SCGB 1A1A proteins form dimers that dissociate under reducing and denaturing conditions. (E) Identity of dimers and monomers was confirmed by Western blot analysis. (C, D, E) S = Precision plus protein standard (dual color).
    Figure Legend Snippet: Cloning, expression and purification of equine recombinant SCGB 1A1 and SCGB 1A1A proteins. (A) SCGB1A1 (“1”) and SCGB1A1A (“1A”) partial ORFs were amplified from equine lung cDNA preparations. A unique band of appropriate size (225 bp) was amplified for each gene. L = 1 Kb+ DNA ladder. (B) Fragments were digested with XmnI and SbfI restriction enzymes (purple boxes) and inserted into the multiple cloning sites (MCS) of the pMAL-c5X expression vector (top). DNA from the transformed colonies was submitted for sequencing to determine the presence, integrity, orientation and suitable translational reading frame of the insert. SCGB1A1 and SCGB1A1A sequenced (S) products showed proper orientation and 100% identity to the predicted (P) sequences. (C) Fractions collected during the purification steps of SCGB 1A1 and SCGB 1A1A were analyzed by SDS-PAGE. A fusion protein was apparent in extracts from IPTG-induced (I) but not un-induced (U) colonies. A crude extract (CE) was collected from induced cells and purified by affinity chromatography, using an amylose (A) column. The eluted fractions were pooled and incubated with Factor Xa protease (Fx) to cleave the fusion proteins. Fx was removed by FPLC (F), and MBP (42.5 kDa) was removed by additional passage on an amylose column from which pure (P) recombinant proteins (7 kDa) were collected. (D) Purified SCGB 1A1 and SCGB 1A1A proteins form dimers that dissociate under reducing and denaturing conditions. (E) Identity of dimers and monomers was confirmed by Western blot analysis. (C, D, E) S = Precision plus protein standard (dual color).

    Techniques Used: Clone Assay, Expressing, Purification, Recombinant, Amplification, Plasmid Preparation, Transformation Assay, Sequencing, SDS Page, Affinity Chromatography, Incubation, Fast Protein Liquid Chromatography, Western Blot

    3) Product Images from "Conserved CxnC motifs in Kaposi’s sarcoma-associated herpesvirus ORF66 are required for viral late gene expression and mediate its interaction with ORF34"

    Article Title: Conserved CxnC motifs in Kaposi’s sarcoma-associated herpesvirus ORF66 are required for viral late gene expression and mediate its interaction with ORF34

    Journal: bioRxiv

    doi: 10.1101/728139

    ORF66 is essential in KSHV and required for late gene transcription A) Diagram showing the genomic locus of ORF66 with surrounding genes ORF67 (which partially overlaps ORF66) and ORF65, depicting the location of introduced mutations. Mutations were confirmed by Sanger sequencing (right). B) Digestion of the recombinant BACs with SbfI or RsrII demonstrates that introduction of mutations did not introduce large-scale changes. C) Infectious virion production was measured by supernatant transfer from reactivated iSLK cell lines followed by flow cytometry. Data are from three independent biological replicates with statistics calculated using an unpaired t-test, where (****) p
    Figure Legend Snippet: ORF66 is essential in KSHV and required for late gene transcription A) Diagram showing the genomic locus of ORF66 with surrounding genes ORF67 (which partially overlaps ORF66) and ORF65, depicting the location of introduced mutations. Mutations were confirmed by Sanger sequencing (right). B) Digestion of the recombinant BACs with SbfI or RsrII demonstrates that introduction of mutations did not introduce large-scale changes. C) Infectious virion production was measured by supernatant transfer from reactivated iSLK cell lines followed by flow cytometry. Data are from three independent biological replicates with statistics calculated using an unpaired t-test, where (****) p

    Techniques Used: Sequencing, Recombinant, Introduce, Flow Cytometry

    ORF24 does not bind to late gene promoters in the absence of ORF30 or ORF66 iSLK cell lines were created using the recombinant BAC16 system. HA tags were added to the endogenous copies of the N-terminus of ORF24 (HA24) or the C-terminus of ORF66 (66HA). In select BACs, ORF24, ORF30, or ORF66 were deleted by the introduction of a stop codon early in the ORF (24S, 30S, and 66S respectively). A) Digestion of the recombinant BACs with SbfI or RsrII demonstrates that recombination did not introduce large-scale changes. B) Infectious virion production was measured by supernatant transfer from reactivated iSLK cell lines followed by flow cytometry. Data are from three independent biological replicates with statistics calculated using an unpaired t-test, where (****) p
    Figure Legend Snippet: ORF24 does not bind to late gene promoters in the absence of ORF30 or ORF66 iSLK cell lines were created using the recombinant BAC16 system. HA tags were added to the endogenous copies of the N-terminus of ORF24 (HA24) or the C-terminus of ORF66 (66HA). In select BACs, ORF24, ORF30, or ORF66 were deleted by the introduction of a stop codon early in the ORF (24S, 30S, and 66S respectively). A) Digestion of the recombinant BACs with SbfI or RsrII demonstrates that recombination did not introduce large-scale changes. B) Infectious virion production was measured by supernatant transfer from reactivated iSLK cell lines followed by flow cytometry. Data are from three independent biological replicates with statistics calculated using an unpaired t-test, where (****) p

    Techniques Used: Recombinant, Introduce, Flow Cytometry

    4) Product Images from "Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)"

    Article Title: Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA118.003302

    Screening for sialidase activity from a hot spring metagenomic library. A , restriction fragment analysis of 12 randomly selected clones from the hot spring metagenomic library were isolated and digested with the rare-cutting endonuclease SbfI. Digested fosmids were separated overnight on a 1% agarose gel along with a λHindIII size marker and a linearized pSMART FOS empty vector control ( lane 14 ; contains one SbfI site). Each clone showed a unique banding pattern, with fragments whose combined sizes indicated the presence of an insert of at least 30–40 kb. B , E. coli cells harboring individual fosmid clones were assayed for sialidase activity with X-Neu5Ac incorporated into agar medium. A single positive clone forming a blue colony is denoted with an arrow. C , lysate from microcultures of E. coli cells harboring individual fosmid clones were assayed for sialidase activity with 4MU-α-Neu5Ac. A single positive clone is denoted with an arrow .
    Figure Legend Snippet: Screening for sialidase activity from a hot spring metagenomic library. A , restriction fragment analysis of 12 randomly selected clones from the hot spring metagenomic library were isolated and digested with the rare-cutting endonuclease SbfI. Digested fosmids were separated overnight on a 1% agarose gel along with a λHindIII size marker and a linearized pSMART FOS empty vector control ( lane 14 ; contains one SbfI site). Each clone showed a unique banding pattern, with fragments whose combined sizes indicated the presence of an insert of at least 30–40 kb. B , E. coli cells harboring individual fosmid clones were assayed for sialidase activity with X-Neu5Ac incorporated into agar medium. A single positive clone forming a blue colony is denoted with an arrow. C , lysate from microcultures of E. coli cells harboring individual fosmid clones were assayed for sialidase activity with 4MU-α-Neu5Ac. A single positive clone is denoted with an arrow .

    Techniques Used: Activity Assay, Clone Assay, Isolation, Agarose Gel Electrophoresis, Marker, Plasmid Preparation

    5) Product Images from "Rapid SNP Discovery and Genetic Mapping Using Sequenced RAD Markers"

    Article Title: Rapid SNP Discovery and Genetic Mapping Using Sequenced RAD Markers

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0003376

    Sequenced RAD marker mapping. (A) A native saltwater stickleback population, Rabbit Slough (RS), have complete lateral plate armor (brackets) while these structures are absent in the derived, freshwater Bear Paw (BP) population. The freshwater fish also have a reduction in pelvic structure (arrow) compared to the oceanic population. These two phenotypes segregate independently in an F 2 mapping cross. Using Sbf I (B) or EcoR I (C), we mapped polymorphic RAD markers from RS (red) and BP (green) parental fish along the 21 stickleback linkage groups. The apparent size differences of the linkage groups between (B) and (C) reflect the fact that the EcoR I recognition sequence occurs more frequently than Sbf I. Red and green bars above the linkage groups are measures of lateral plate linkage in the F 2 progeny, indicating the number of tightly linked markers in the local region. (D) Sequence reads per barcoded F 2 individual used to create (C). Variable numbers of reads were obtained from each of the 96 individuals used in our analysis, reflecting different concentrations of starting DNA template. 68% of individuals had between 50 K and 150 K RAD tags sequenced (∼0.4–1.0× coverage of the ∼150 K tags present in the genome). Only 2 individuals had less than 10,000 reads (red). (E) A close-up of the boxed region from (C) showing recombination breakpoints in six informative low plate F 2 fish on LGIV. Black tick marks are 1 Mb apart in physical distance. (F) F 2 individuals were repooled in silico based on the pelvic structure phenotype (A, arrow). Linkage was determined as in (B, C), mapping the locus for a reduction in pelvic structure to the end of LGVII.
    Figure Legend Snippet: Sequenced RAD marker mapping. (A) A native saltwater stickleback population, Rabbit Slough (RS), have complete lateral plate armor (brackets) while these structures are absent in the derived, freshwater Bear Paw (BP) population. The freshwater fish also have a reduction in pelvic structure (arrow) compared to the oceanic population. These two phenotypes segregate independently in an F 2 mapping cross. Using Sbf I (B) or EcoR I (C), we mapped polymorphic RAD markers from RS (red) and BP (green) parental fish along the 21 stickleback linkage groups. The apparent size differences of the linkage groups between (B) and (C) reflect the fact that the EcoR I recognition sequence occurs more frequently than Sbf I. Red and green bars above the linkage groups are measures of lateral plate linkage in the F 2 progeny, indicating the number of tightly linked markers in the local region. (D) Sequence reads per barcoded F 2 individual used to create (C). Variable numbers of reads were obtained from each of the 96 individuals used in our analysis, reflecting different concentrations of starting DNA template. 68% of individuals had between 50 K and 150 K RAD tags sequenced (∼0.4–1.0× coverage of the ∼150 K tags present in the genome). Only 2 individuals had less than 10,000 reads (red). (E) A close-up of the boxed region from (C) showing recombination breakpoints in six informative low plate F 2 fish on LGIV. Black tick marks are 1 Mb apart in physical distance. (F) F 2 individuals were repooled in silico based on the pelvic structure phenotype (A, arrow). Linkage was determined as in (B, C), mapping the locus for a reduction in pelvic structure to the end of LGVII.

    Techniques Used: Marker, Derivative Assay, Fluorescence In Situ Hybridization, Sequencing, In Silico

    6) Product Images from "A pentameric protein ring with novel architecture is required for herpesviral packaging"

    Article Title: A pentameric protein ring with novel architecture is required for herpesviral packaging

    Journal: bioRxiv

    doi: 10.1101/2020.07.16.206755

    Construction and validation of mutant viruses. a , Schematic of the genomic locus of ORF68, with the location of introduced mutations depicted in detail below. Sanger sequencing traces for the mutants and corresponding mutant rescues are shown to the right. b , Digestion of recombinant BACs with RsrII and SbfI was used to assess whether large-scale recombination had occurred during mutagenesis. c , Western blot of whole cell lysate (25 μg) from ORF68.stop iSLK cell lines. GAPDH was used as a loading control. ORF6 is an early gene and K8.1 is a late gene. d , Viral DNA replication was measured by qPCR before and after reactivation. Data are from three independent biological replicates, with statistics being calculated using an unpaired t test. **, p
    Figure Legend Snippet: Construction and validation of mutant viruses. a , Schematic of the genomic locus of ORF68, with the location of introduced mutations depicted in detail below. Sanger sequencing traces for the mutants and corresponding mutant rescues are shown to the right. b , Digestion of recombinant BACs with RsrII and SbfI was used to assess whether large-scale recombination had occurred during mutagenesis. c , Western blot of whole cell lysate (25 μg) from ORF68.stop iSLK cell lines. GAPDH was used as a loading control. ORF6 is an early gene and K8.1 is a late gene. d , Viral DNA replication was measured by qPCR before and after reactivation. Data are from three independent biological replicates, with statistics being calculated using an unpaired t test. **, p

    Techniques Used: Mutagenesis, Sequencing, Recombinant, Western Blot, Real-time Polymerase Chain Reaction

    7) Product Images from "Characterization of the spore-forming Bacillus cereus sensu lato group and Clostridium perfringens bacteria isolated from the Australian dairy farm environment"

    Article Title: Characterization of the spore-forming Bacillus cereus sensu lato group and Clostridium perfringens bacteria isolated from the Australian dairy farm environment

    Journal: BMC Microbiology

    doi: 10.1186/s12866-015-0377-9

    Dendogram of the PFGE profiles of the B. cereus group isolates, using combined Not I and Sbf I restriction analysis. a +, growth; -, no growth. b S, strong, W, weak.
    Figure Legend Snippet: Dendogram of the PFGE profiles of the B. cereus group isolates, using combined Not I and Sbf I restriction analysis. a +, growth; -, no growth. b S, strong, W, weak.

    Techniques Used:

    8) Product Images from "Amplification Biases and Consistent Recovery of Loci in a Double-Digest RAD-seq Protocol"

    Article Title: Amplification Biases and Consistent Recovery of Loci in a Double-Digest RAD-seq Protocol

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0106713

    Sequencing depth for single copy ddRAD loci in relation to the corresponding sequence in the zebra finch reference genome. Categories from top to bottom include: loci mapping as expected to predicted SbfI-EcoRI restriction fragments≤328 bp in length; all loci beginning at a genomic location similar but not identical to the canonical SbfI recognition sequence (1–4 mismatches); subset of loci with one mismatch in position 1 or 8 of the SbfI recognition sequence; subset of loci with one mismatch in positions 2 through 7 of the SbfI recognition sequence; loci mapping to a genomic SbfI site without an EcoRI site within 328 bp; and loci mapping to a predicted SbfI-SbfI restriction fragment less than 328 bp in length.
    Figure Legend Snippet: Sequencing depth for single copy ddRAD loci in relation to the corresponding sequence in the zebra finch reference genome. Categories from top to bottom include: loci mapping as expected to predicted SbfI-EcoRI restriction fragments≤328 bp in length; all loci beginning at a genomic location similar but not identical to the canonical SbfI recognition sequence (1–4 mismatches); subset of loci with one mismatch in position 1 or 8 of the SbfI recognition sequence; subset of loci with one mismatch in positions 2 through 7 of the SbfI recognition sequence; loci mapping to a genomic SbfI site without an EcoRI site within 328 bp; and loci mapping to a predicted SbfI-SbfI restriction fragment less than 328 bp in length.

    Techniques Used: Sequencing

    Recovery and sequencing depth for predicted, single-copy ddRAD loci in the empirical zebra finch data. (A) Proportion of predicted loci recovered at three different minimum depth thresholds as a function of predicted fragment length. Each data point represents the proportion of ∼140–220 predicted loci recovered in a given 10 bp size range. Dashed vertical lines represent the upper and lower bounds of the size range isolated from the agarose gel. (B) Sequencing depth for recovered (depth ≥1), single-copy loci in the 32–500 bp size range (includes 5,232 of 5,783 predicted loci in the 38–328 bp size range). (C) The relationship between GC content and sequencing depth for zebra finch ddRAD loci. Data are shown for predicted, single-copy loci recovered at a depth ≥1 in three selected subsets of the overall size range ( n = 502, 466, and 445 loci in the 100–125, 200–225, and 300–325 bp size ranges, respectively). The predicted length and GC content of each locus are based on the full-length fragment in the reference genome, inclusive of the SbfI and EcoRI restriction sites on either end. Note that the y-axis is on a logarithmic scale in (B) and (C).
    Figure Legend Snippet: Recovery and sequencing depth for predicted, single-copy ddRAD loci in the empirical zebra finch data. (A) Proportion of predicted loci recovered at three different minimum depth thresholds as a function of predicted fragment length. Each data point represents the proportion of ∼140–220 predicted loci recovered in a given 10 bp size range. Dashed vertical lines represent the upper and lower bounds of the size range isolated from the agarose gel. (B) Sequencing depth for recovered (depth ≥1), single-copy loci in the 32–500 bp size range (includes 5,232 of 5,783 predicted loci in the 38–328 bp size range). (C) The relationship between GC content and sequencing depth for zebra finch ddRAD loci. Data are shown for predicted, single-copy loci recovered at a depth ≥1 in three selected subsets of the overall size range ( n = 502, 466, and 445 loci in the 100–125, 200–225, and 300–325 bp size ranges, respectively). The predicted length and GC content of each locus are based on the full-length fragment in the reference genome, inclusive of the SbfI and EcoRI restriction sites on either end. Note that the y-axis is on a logarithmic scale in (B) and (C).

    Techniques Used: Sequencing, Isolation, Agarose Gel Electrophoresis

    9) Product Images from "Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)"

    Article Title: Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA118.003302

    Screening for sialidase activity from a hot spring metagenomic library. A , restriction fragment analysis of 12 randomly selected clones from the hot spring metagenomic library were isolated and digested with the rare-cutting endonuclease SbfI. Digested fosmids were separated overnight on a 1% agarose gel along with a λHindIII size marker and a linearized pSMART FOS empty vector control ( lane 14 ; contains one SbfI site). Each clone showed a unique banding pattern, with fragments whose combined sizes indicated the presence of an insert of at least 30–40 kb. B , E. coli cells harboring individual fosmid clones were assayed for sialidase activity with X-Neu5Ac incorporated into agar medium. A single positive clone forming a blue colony is denoted with an arrow. C , lysate from microcultures of E. coli cells harboring individual fosmid clones were assayed for sialidase activity with 4MU-α-Neu5Ac. A single positive clone is denoted with an arrow .
    Figure Legend Snippet: Screening for sialidase activity from a hot spring metagenomic library. A , restriction fragment analysis of 12 randomly selected clones from the hot spring metagenomic library were isolated and digested with the rare-cutting endonuclease SbfI. Digested fosmids were separated overnight on a 1% agarose gel along with a λHindIII size marker and a linearized pSMART FOS empty vector control ( lane 14 ; contains one SbfI site). Each clone showed a unique banding pattern, with fragments whose combined sizes indicated the presence of an insert of at least 30–40 kb. B , E. coli cells harboring individual fosmid clones were assayed for sialidase activity with X-Neu5Ac incorporated into agar medium. A single positive clone forming a blue colony is denoted with an arrow. C , lysate from microcultures of E. coli cells harboring individual fosmid clones were assayed for sialidase activity with 4MU-α-Neu5Ac. A single positive clone is denoted with an arrow .

    Techniques Used: Activity Assay, Clone Assay, Isolation, Agarose Gel Electrophoresis, Marker, Plasmid Preparation

    10) Product Images from "Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)"

    Article Title: Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA118.003302

    Screening for sialidase activity from a hot spring metagenomic library. A , restriction fragment analysis of 12 randomly selected clones from the hot spring metagenomic library were isolated and digested with the rare-cutting endonuclease SbfI. Digested fosmids were separated overnight on a 1% agarose gel along with a λHindIII size marker and a linearized pSMART FOS empty vector control ( lane 14 ; contains one SbfI site). Each clone showed a unique banding pattern, with fragments whose combined sizes indicated the presence of an insert of at least 30–40 kb. B , E. coli cells harboring individual fosmid clones were assayed for sialidase activity with X-Neu5Ac incorporated into agar medium. A single positive clone forming a blue colony is denoted with an arrow. C , lysate from microcultures of E. coli cells harboring individual fosmid clones were assayed for sialidase activity with 4MU-α-Neu5Ac. A single positive clone is denoted with an arrow .
    Figure Legend Snippet: Screening for sialidase activity from a hot spring metagenomic library. A , restriction fragment analysis of 12 randomly selected clones from the hot spring metagenomic library were isolated and digested with the rare-cutting endonuclease SbfI. Digested fosmids were separated overnight on a 1% agarose gel along with a λHindIII size marker and a linearized pSMART FOS empty vector control ( lane 14 ; contains one SbfI site). Each clone showed a unique banding pattern, with fragments whose combined sizes indicated the presence of an insert of at least 30–40 kb. B , E. coli cells harboring individual fosmid clones were assayed for sialidase activity with X-Neu5Ac incorporated into agar medium. A single positive clone forming a blue colony is denoted with an arrow. C , lysate from microcultures of E. coli cells harboring individual fosmid clones were assayed for sialidase activity with 4MU-α-Neu5Ac. A single positive clone is denoted with an arrow .

    Techniques Used: Activity Assay, Clone Assay, Isolation, Agarose Gel Electrophoresis, Marker, Plasmid Preparation

    Related Articles

    Clone Assay:

    Article Title: Secretoglobin 1A1 and 1A1A Differentially Regulate Neutrophil Reactive Oxygen Species Production, Phagocytosis and Extracellular Trap Formation
    Article Snippet: .. Primers were designed with XmnI and SbfI restriction sites for cloning into the pMAL-c5X expression vector (New England BioLabs, Mississauga, ON). .. This vector is designed to produce maltose-binding protein (MBP) fusion proteins without adding vector-derived residues.

    BAC Assay:

    Article Title: A pentameric protein ring with novel architecture is required for herpesviral packaging
    Article Snippet: .. BAC quality was assessed by digestion with RsrII and SbfI (New England Biolabs). .. Latently infected iSLK cell lines with modified virus were generated by transfection of HEK293T-ORF68 cells with 5 μg BAC DNA using PolyJet reagent (SignaGen).

    Article Title: Conserved CxnC Motifs in Kaposi’s Sarcoma-Associated Herpesvirus ORF66 Are Required for Viral Late Gene Expression and Are Essential for Its Interaction with ORF34
    Article Snippet: .. BAC quality was assessed by digestion with RsrII and SbfI (New England Biolabs). .. Latently infected iSLK cell lines with modified virus were generated by transfection of HEK293T cells (either WT cells or cells stably expressing the relevant essential viral ORF) with 5 μg BAC DNA using the PolyJet reagent (SignaGen).

    Article Title: Conserved CxnC motifs in Kaposi’s sarcoma-associated herpesvirus ORF66 are required for viral late gene expression and mediate its interaction with ORF34
    Article Snippet: .. BAC quality was assessed by digestion with RsrII and SbfI (New England Biolabs). .. Latently infected iSLK cell lines with modified virus were generated by transfection of HEK293T cells (either WT or stably expressing the relevant essential viral ORF) with 5 μg BAC DNA using PolyJet (SignaGen).

    Isolation:

    Article Title: Rapid SNP Discovery and Genetic Mapping Using Sequenced RAD Markers
    Article Snippet: .. Isolation of RAD markers for Illumina sequencing Genomic DNA (0.1–1 µg; from either individual or pooled samples) was digested for 15 min at 37°C in a 50 µL reaction with 20 units (U) of EcoR I or Sbf I (New England Biolabs [NEB]). ..

    Expressing:

    Article Title: Secretoglobin 1A1 and 1A1A Differentially Regulate Neutrophil Reactive Oxygen Species Production, Phagocytosis and Extracellular Trap Formation
    Article Snippet: .. Primers were designed with XmnI and SbfI restriction sites for cloning into the pMAL-c5X expression vector (New England BioLabs, Mississauga, ON). .. This vector is designed to produce maltose-binding protein (MBP) fusion proteins without adding vector-derived residues.

    Sequencing:

    Article Title: Rapid SNP Discovery and Genetic Mapping Using Sequenced RAD Markers
    Article Snippet: .. Isolation of RAD markers for Illumina sequencing Genomic DNA (0.1–1 µg; from either individual or pooled samples) was digested for 15 min at 37°C in a 50 µL reaction with 20 units (U) of EcoR I or Sbf I (New England Biolabs [NEB]). ..

    Plasmid Preparation:

    Article Title: Secretoglobin 1A1 and 1A1A Differentially Regulate Neutrophil Reactive Oxygen Species Production, Phagocytosis and Extracellular Trap Formation
    Article Snippet: .. Primers were designed with XmnI and SbfI restriction sites for cloning into the pMAL-c5X expression vector (New England BioLabs, Mississauga, ON). .. This vector is designed to produce maltose-binding protein (MBP) fusion proteins without adding vector-derived residues.

    Article Title: Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)
    Article Snippet: .. Each fosmid was digested with SbfI (New England Biolabs), a unique restriction site in the pSMART FOS vector. ..

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    New England Biolabs sbfi
    ORF66 is essential in KSHV and required for late gene transcription. (A) (Left) Diagram showing the genomic locus of ORF66 with surrounding genes ORF67 (which partially overlaps ORF66) and ORF65, depicting the location of introduced mutations. (Right) The mutations were confirmed by Sanger sequencing. (B) Digestion of the recombinant BACs with <t>SbfI</t> or <t>RsrII</t> demonstrates that the introduction of mutations did not introduce large-scale changes. (C) Infectious virion production was measured by supernatant transfer from reactivated iSLK cell lines followed by flow cytometry. Data are from three independent biological replicates, with statistics being calculated using an unpaired t test. ****, P
    Sbfi, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ORF66 is essential in KSHV and required for late gene transcription. (A) (Left) Diagram showing the genomic locus of ORF66 with surrounding genes ORF67 (which partially overlaps ORF66) and ORF65, depicting the location of introduced mutations. (Right) The mutations were confirmed by Sanger sequencing. (B) Digestion of the recombinant BACs with SbfI or RsrII demonstrates that the introduction of mutations did not introduce large-scale changes. (C) Infectious virion production was measured by supernatant transfer from reactivated iSLK cell lines followed by flow cytometry. Data are from three independent biological replicates, with statistics being calculated using an unpaired t test. ****, P

    Journal: Journal of Virology

    Article Title: Conserved CxnC Motifs in Kaposi’s Sarcoma-Associated Herpesvirus ORF66 Are Required for Viral Late Gene Expression and Are Essential for Its Interaction with ORF34

    doi: 10.1128/JVI.01299-19

    Figure Lengend Snippet: ORF66 is essential in KSHV and required for late gene transcription. (A) (Left) Diagram showing the genomic locus of ORF66 with surrounding genes ORF67 (which partially overlaps ORF66) and ORF65, depicting the location of introduced mutations. (Right) The mutations were confirmed by Sanger sequencing. (B) Digestion of the recombinant BACs with SbfI or RsrII demonstrates that the introduction of mutations did not introduce large-scale changes. (C) Infectious virion production was measured by supernatant transfer from reactivated iSLK cell lines followed by flow cytometry. Data are from three independent biological replicates, with statistics being calculated using an unpaired t test. ****, P

    Article Snippet: BAC quality was assessed by digestion with RsrII and SbfI (New England Biolabs).

    Techniques: Sequencing, Recombinant, Introduce, Flow Cytometry

    Cloning, expression and purification of equine recombinant SCGB 1A1 and SCGB 1A1A proteins. (A) SCGB1A1 (“1”) and SCGB1A1A (“1A”) partial ORFs were amplified from equine lung cDNA preparations. A unique band of appropriate size (225 bp) was amplified for each gene. L = 1 Kb+ DNA ladder. (B) Fragments were digested with XmnI and SbfI restriction enzymes (purple boxes) and inserted into the multiple cloning sites (MCS) of the pMAL-c5X expression vector (top). DNA from the transformed colonies was submitted for sequencing to determine the presence, integrity, orientation and suitable translational reading frame of the insert. SCGB1A1 and SCGB1A1A sequenced (S) products showed proper orientation and 100% identity to the predicted (P) sequences. (C) Fractions collected during the purification steps of SCGB 1A1 and SCGB 1A1A were analyzed by SDS-PAGE. A fusion protein was apparent in extracts from IPTG-induced (I) but not un-induced (U) colonies. A crude extract (CE) was collected from induced cells and purified by affinity chromatography, using an amylose (A) column. The eluted fractions were pooled and incubated with Factor Xa protease (Fx) to cleave the fusion proteins. Fx was removed by FPLC (F), and MBP (42.5 kDa) was removed by additional passage on an amylose column from which pure (P) recombinant proteins (7 kDa) were collected. (D) Purified SCGB 1A1 and SCGB 1A1A proteins form dimers that dissociate under reducing and denaturing conditions. (E) Identity of dimers and monomers was confirmed by Western blot analysis. (C, D, E) S = Precision plus protein standard (dual color).

    Journal: PLoS ONE

    Article Title: Secretoglobin 1A1 and 1A1A Differentially Regulate Neutrophil Reactive Oxygen Species Production, Phagocytosis and Extracellular Trap Formation

    doi: 10.1371/journal.pone.0096217

    Figure Lengend Snippet: Cloning, expression and purification of equine recombinant SCGB 1A1 and SCGB 1A1A proteins. (A) SCGB1A1 (“1”) and SCGB1A1A (“1A”) partial ORFs were amplified from equine lung cDNA preparations. A unique band of appropriate size (225 bp) was amplified for each gene. L = 1 Kb+ DNA ladder. (B) Fragments were digested with XmnI and SbfI restriction enzymes (purple boxes) and inserted into the multiple cloning sites (MCS) of the pMAL-c5X expression vector (top). DNA from the transformed colonies was submitted for sequencing to determine the presence, integrity, orientation and suitable translational reading frame of the insert. SCGB1A1 and SCGB1A1A sequenced (S) products showed proper orientation and 100% identity to the predicted (P) sequences. (C) Fractions collected during the purification steps of SCGB 1A1 and SCGB 1A1A were analyzed by SDS-PAGE. A fusion protein was apparent in extracts from IPTG-induced (I) but not un-induced (U) colonies. A crude extract (CE) was collected from induced cells and purified by affinity chromatography, using an amylose (A) column. The eluted fractions were pooled and incubated with Factor Xa protease (Fx) to cleave the fusion proteins. Fx was removed by FPLC (F), and MBP (42.5 kDa) was removed by additional passage on an amylose column from which pure (P) recombinant proteins (7 kDa) were collected. (D) Purified SCGB 1A1 and SCGB 1A1A proteins form dimers that dissociate under reducing and denaturing conditions. (E) Identity of dimers and monomers was confirmed by Western blot analysis. (C, D, E) S = Precision plus protein standard (dual color).

    Article Snippet: Primers were designed with XmnI and SbfI restriction sites for cloning into the pMAL-c5X expression vector (New England BioLabs, Mississauga, ON).

    Techniques: Clone Assay, Expressing, Purification, Recombinant, Amplification, Plasmid Preparation, Transformation Assay, Sequencing, SDS Page, Affinity Chromatography, Incubation, Fast Protein Liquid Chromatography, Western Blot

    ORF66 is essential in KSHV and required for late gene transcription A) Diagram showing the genomic locus of ORF66 with surrounding genes ORF67 (which partially overlaps ORF66) and ORF65, depicting the location of introduced mutations. Mutations were confirmed by Sanger sequencing (right). B) Digestion of the recombinant BACs with SbfI or RsrII demonstrates that introduction of mutations did not introduce large-scale changes. C) Infectious virion production was measured by supernatant transfer from reactivated iSLK cell lines followed by flow cytometry. Data are from three independent biological replicates with statistics calculated using an unpaired t-test, where (****) p

    Journal: bioRxiv

    Article Title: Conserved CxnC motifs in Kaposi’s sarcoma-associated herpesvirus ORF66 are required for viral late gene expression and mediate its interaction with ORF34

    doi: 10.1101/728139

    Figure Lengend Snippet: ORF66 is essential in KSHV and required for late gene transcription A) Diagram showing the genomic locus of ORF66 with surrounding genes ORF67 (which partially overlaps ORF66) and ORF65, depicting the location of introduced mutations. Mutations were confirmed by Sanger sequencing (right). B) Digestion of the recombinant BACs with SbfI or RsrII demonstrates that introduction of mutations did not introduce large-scale changes. C) Infectious virion production was measured by supernatant transfer from reactivated iSLK cell lines followed by flow cytometry. Data are from three independent biological replicates with statistics calculated using an unpaired t-test, where (****) p

    Article Snippet: BAC quality was assessed by digestion with RsrII and SbfI (New England Biolabs).

    Techniques: Sequencing, Recombinant, Introduce, Flow Cytometry

    ORF24 does not bind to late gene promoters in the absence of ORF30 or ORF66 iSLK cell lines were created using the recombinant BAC16 system. HA tags were added to the endogenous copies of the N-terminus of ORF24 (HA24) or the C-terminus of ORF66 (66HA). In select BACs, ORF24, ORF30, or ORF66 were deleted by the introduction of a stop codon early in the ORF (24S, 30S, and 66S respectively). A) Digestion of the recombinant BACs with SbfI or RsrII demonstrates that recombination did not introduce large-scale changes. B) Infectious virion production was measured by supernatant transfer from reactivated iSLK cell lines followed by flow cytometry. Data are from three independent biological replicates with statistics calculated using an unpaired t-test, where (****) p

    Journal: bioRxiv

    Article Title: Conserved CxnC motifs in Kaposi’s sarcoma-associated herpesvirus ORF66 are required for viral late gene expression and mediate its interaction with ORF34

    doi: 10.1101/728139

    Figure Lengend Snippet: ORF24 does not bind to late gene promoters in the absence of ORF30 or ORF66 iSLK cell lines were created using the recombinant BAC16 system. HA tags were added to the endogenous copies of the N-terminus of ORF24 (HA24) or the C-terminus of ORF66 (66HA). In select BACs, ORF24, ORF30, or ORF66 were deleted by the introduction of a stop codon early in the ORF (24S, 30S, and 66S respectively). A) Digestion of the recombinant BACs with SbfI or RsrII demonstrates that recombination did not introduce large-scale changes. B) Infectious virion production was measured by supernatant transfer from reactivated iSLK cell lines followed by flow cytometry. Data are from three independent biological replicates with statistics calculated using an unpaired t-test, where (****) p

    Article Snippet: BAC quality was assessed by digestion with RsrII and SbfI (New England Biolabs).

    Techniques: Recombinant, Introduce, Flow Cytometry

    Screening for sialidase activity from a hot spring metagenomic library. A , restriction fragment analysis of 12 randomly selected clones from the hot spring metagenomic library were isolated and digested with the rare-cutting endonuclease SbfI. Digested fosmids were separated overnight on a 1% agarose gel along with a λHindIII size marker and a linearized pSMART FOS empty vector control ( lane 14 ; contains one SbfI site). Each clone showed a unique banding pattern, with fragments whose combined sizes indicated the presence of an insert of at least 30–40 kb. B , E. coli cells harboring individual fosmid clones were assayed for sialidase activity with X-Neu5Ac incorporated into agar medium. A single positive clone forming a blue colony is denoted with an arrow. C , lysate from microcultures of E. coli cells harboring individual fosmid clones were assayed for sialidase activity with 4MU-α-Neu5Ac. A single positive clone is denoted with an arrow .

    Journal: The Journal of Biological Chemistry

    Article Title: Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)

    doi: 10.1074/jbc.RA118.003302

    Figure Lengend Snippet: Screening for sialidase activity from a hot spring metagenomic library. A , restriction fragment analysis of 12 randomly selected clones from the hot spring metagenomic library were isolated and digested with the rare-cutting endonuclease SbfI. Digested fosmids were separated overnight on a 1% agarose gel along with a λHindIII size marker and a linearized pSMART FOS empty vector control ( lane 14 ; contains one SbfI site). Each clone showed a unique banding pattern, with fragments whose combined sizes indicated the presence of an insert of at least 30–40 kb. B , E. coli cells harboring individual fosmid clones were assayed for sialidase activity with X-Neu5Ac incorporated into agar medium. A single positive clone forming a blue colony is denoted with an arrow. C , lysate from microcultures of E. coli cells harboring individual fosmid clones were assayed for sialidase activity with 4MU-α-Neu5Ac. A single positive clone is denoted with an arrow .

    Article Snippet: Each fosmid was digested with SbfI (New England Biolabs), a unique restriction site in the pSMART FOS vector.

    Techniques: Activity Assay, Clone Assay, Isolation, Agarose Gel Electrophoresis, Marker, Plasmid Preparation