sspi hf  (New England Biolabs)


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  • 90
    Name:
    SspI HF
    Description:
    SspI HF 5 000 units
    Catalog Number:
    r3132l
    Price:
    285
    Size:
    5 000 units
    Category:
    Restriction Enzymes
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    Structured Review

    New England Biolabs sspi hf
    SspI HF
    SspI HF 5 000 units
    https://www.bioz.com/result/sspi hf/product/New England Biolabs
    Average 90 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    sspi hf - by Bioz Stars, 2020-01
    90/100 stars

    Images

    1) Product Images from "Fallacy of the Unique Genome: Sequence Diversity within Single Helicobacter pylori Strains"

    Article Title: Fallacy of the Unique Genome: Sequence Diversity within Single Helicobacter pylori Strains

    Journal: mBio

    doi: 10.1128/mBio.02321-16

    H. pylori SS1 and PMSS1 are gene copy number variable at the cagA locus. (A) Schematic diagram showing tandem arrays of the identical 5,072-bp repeat regions at the cagA locus, all of which contain identical copies of the 3,540-bp cagA gene. The SspI fragment sizes of strains with 1 to 4 cagA copies are shown at the right. The drawing is not to scale. (B) Southern blot of SspI-digested genomic DNA from H. pylori SS1 or PMSS1 probed with a 297-bp PCR product amplified from SS1 cagA bp 1217 to 1514. The original working stocks of SS1 (lane 1) and PMSS1 (lane 5) showed bands corresponding in size to 4, 3, 2, and 1 copies of cagA (asterisks). Subculture of 4 single colonies from the freezer stocks demonstrated clones with 4, 3, or 2 copies of cagA (lanes 2 to 4) from SS1; subculture of 12 single colonies showed either 4 or 2 copies of cagA in PMSS1 (lanes 6 to 8). PMSS1 with a cagA deletion served as a negative control (lane 9). A kilobase ladder is shown at the left. (C) Western blot of H. pylori PMSS1 to examine whether the cagA copy number is positively correlated with protein expression. For this analysis, six individual single-colony isolates of PMSS1 were used, two each with four copies, two copies, or one copy of cagA . Relative quantities of protein in each band were determined using Image Lab software (Bio-Rad Laboratories). The density of the CagA band was divided by the density of the corresponding UreB band to obtain the normalized quantity of CagA to account for differences in gel loading.
    Figure Legend Snippet: H. pylori SS1 and PMSS1 are gene copy number variable at the cagA locus. (A) Schematic diagram showing tandem arrays of the identical 5,072-bp repeat regions at the cagA locus, all of which contain identical copies of the 3,540-bp cagA gene. The SspI fragment sizes of strains with 1 to 4 cagA copies are shown at the right. The drawing is not to scale. (B) Southern blot of SspI-digested genomic DNA from H. pylori SS1 or PMSS1 probed with a 297-bp PCR product amplified from SS1 cagA bp 1217 to 1514. The original working stocks of SS1 (lane 1) and PMSS1 (lane 5) showed bands corresponding in size to 4, 3, 2, and 1 copies of cagA (asterisks). Subculture of 4 single colonies from the freezer stocks demonstrated clones with 4, 3, or 2 copies of cagA (lanes 2 to 4) from SS1; subculture of 12 single colonies showed either 4 or 2 copies of cagA in PMSS1 (lanes 6 to 8). PMSS1 with a cagA deletion served as a negative control (lane 9). A kilobase ladder is shown at the left. (C) Western blot of H. pylori PMSS1 to examine whether the cagA copy number is positively correlated with protein expression. For this analysis, six individual single-colony isolates of PMSS1 were used, two each with four copies, two copies, or one copy of cagA . Relative quantities of protein in each band were determined using Image Lab software (Bio-Rad Laboratories). The density of the CagA band was divided by the density of the corresponding UreB band to obtain the normalized quantity of CagA to account for differences in gel loading.

    Techniques Used: Southern Blot, Polymerase Chain Reaction, Amplification, Clone Assay, Negative Control, Western Blot, Expressing, Software

    2) Product Images from "The HIV Protease Inhibitor Nelfinavir Inhibits Kaposi's Sarcoma-Associated Herpesvirus Replication In Vitro ▿"

    Article Title: The HIV Protease Inhibitor Nelfinavir Inhibits Kaposi's Sarcoma-Associated Herpesvirus Replication In Vitro ▿

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.01295-10

    Construction and quantitation of rKSHV.294. (A) Schematic diagram of rKSHV.294 showing the insertion site in the KSHV genome; the BamHI sites flanking the 4.8-kb segment of the KSHV genome used are indicated. The relative positions of the SeAP, the GFP, and the Neo elements are shown with their respective promoters but not to scale. Beneath the 294 construct are shown the expected PCR products (a to e) with the primers used for analysis of viral DNA. (B) Hybridization analysis of rKSHV.294 and JSC-1 viral DNA. (Left) KSHV probe using the 4.8-kb BamHI fragment used to construct the virus. (Right) Neo probe. Lane 1, rKSHV.294 × AflII, predicted fragments of 15.9, 5.7, and 4.8 kb. The 4.8-kb fragment contains the SeAP/GFP/Neo insert with only 170 bp of KSHV DNA, which accounts for the weak band. Lane 2, JSC-1 × AflII, predicted fragments of 15.9 and 5.8 kb. Lane 3, rKSHV.294 × SspI, predicted fragments of 8 and 6.2 kb. Lane 4, JSC-1 × SspI, predicted fragments of 6.2 and 3.3 kb. Lane 5, rKSHV.294 × AflII, predicted fragment of 4.8 kb. Lane 6, JSC-1 × AflII. Lane 7, rKSHV.294 DNA × SspI, predicted fragment of 8 kb. Lane 8, JSC-1 × SspI. DNA markers in kb are on the left side. (C) Ethidium bromide-stained agarose gel following electrophoresis of the PCR products resulting with rKSHV.294 viral DNA with the indicated primers described in Materials and Methods. Lane a, ORF56f (outside BamHI site) and SeAPr; lane b, ORF57u and SeAPr; lane c, SeAPf and GFPf; lane d, GFPr and Neor; lane e, Neof and K9. (D) rKSHV.294 was tested for tTA control of SeAP expression by the infection of 293 cells or of 293 cells expressing tTA at an MOI of
    Figure Legend Snippet: Construction and quantitation of rKSHV.294. (A) Schematic diagram of rKSHV.294 showing the insertion site in the KSHV genome; the BamHI sites flanking the 4.8-kb segment of the KSHV genome used are indicated. The relative positions of the SeAP, the GFP, and the Neo elements are shown with their respective promoters but not to scale. Beneath the 294 construct are shown the expected PCR products (a to e) with the primers used for analysis of viral DNA. (B) Hybridization analysis of rKSHV.294 and JSC-1 viral DNA. (Left) KSHV probe using the 4.8-kb BamHI fragment used to construct the virus. (Right) Neo probe. Lane 1, rKSHV.294 × AflII, predicted fragments of 15.9, 5.7, and 4.8 kb. The 4.8-kb fragment contains the SeAP/GFP/Neo insert with only 170 bp of KSHV DNA, which accounts for the weak band. Lane 2, JSC-1 × AflII, predicted fragments of 15.9 and 5.8 kb. Lane 3, rKSHV.294 × SspI, predicted fragments of 8 and 6.2 kb. Lane 4, JSC-1 × SspI, predicted fragments of 6.2 and 3.3 kb. Lane 5, rKSHV.294 × AflII, predicted fragment of 4.8 kb. Lane 6, JSC-1 × AflII. Lane 7, rKSHV.294 DNA × SspI, predicted fragment of 8 kb. Lane 8, JSC-1 × SspI. DNA markers in kb are on the left side. (C) Ethidium bromide-stained agarose gel following electrophoresis of the PCR products resulting with rKSHV.294 viral DNA with the indicated primers described in Materials and Methods. Lane a, ORF56f (outside BamHI site) and SeAPr; lane b, ORF57u and SeAPr; lane c, SeAPf and GFPf; lane d, GFPr and Neor; lane e, Neof and K9. (D) rKSHV.294 was tested for tTA control of SeAP expression by the infection of 293 cells or of 293 cells expressing tTA at an MOI of

    Techniques Used: Quantitation Assay, Construct, Polymerase Chain Reaction, Hybridization, Staining, Agarose Gel Electrophoresis, Electrophoresis, Expressing, Infection

    Related Articles

    Amplification:

    Article Title: Fallacy of the Unique Genome: Sequence Diversity within Single Helicobacter pylori Strains
    Article Snippet: Genomic DNA was digested with SspI-HF (New England Biolabs) for 2 h. Digested DNA was separated on a 0.5% agarose gel overnight at 0.75 V/cm and transferred to a nylon membrane. .. A fragment of cagA was PCR amplified from SS1 (bp 1217 to 1514) with primers D008 and R008 ( ) and labeled with biotin using a North2South biotin Random Prime Labeling kit (Thermo Scientific).

    Expressing:

    Article Title: Fallacy of the Unique Genome: Sequence Diversity within Single Helicobacter pylori Strains
    Article Snippet: Paragraph title: cagA copy number verification and CagA expression analysis. ... Genomic DNA was digested with SspI-HF (New England Biolabs) for 2 h. Digested DNA was separated on a 0.5% agarose gel overnight at 0.75 V/cm and transferred to a nylon membrane.

    Electrophoresis:

    Article Title: In vitro Enzymology of Cas9
    Article Snippet: For pUC19-based plasmids, use 50 units of SspI-HF (New England Biolabs) and 5 μg plasmid in 1 x CutSmart™ buffer in a total volume of 50 μl. .. Analyze plasmid cleavage by electrophoresis on a 1 % agarose gel in 1 x TAE buffer (40 mM Tris, pH 8.0, 20 mM glacial acetic acid, 1 mM EDTA, pH 8.0) stained with GelRed (Biotium) or similar nucleic acid stain.

    Incubation:

    Article Title: Comparison of diets for Largemouth and Smallmouth Bass in Eastern Lake Ontario using DNA barcoding and stable isotope analysis
    Article Snippet: .. Restriction digest of fish prey PCRs occurred in 12.5 μl reactions consisting of; 5 μl PCR product; 1x CutSmart Buffer; and 1 U SspI-HF (New England Biolabs), incubated for 30 min at 37°C. .. PCR and restriction digest success was evaluated by running total digest volumes in 2% agarose gels.

    Article Title: Structural basis for guide RNA processing and seed-dependent DNA targeting and cleavage by CRISPR-Cas12a
    Article Snippet: Plasmids were linearized by incubating 640 ng of plasmid DNA with 15 units of SspI-HF (New England Biolabs) in CutSmart buffer (New England Biolabs) for 1 h at 37 °C, followed by heat-inactivation for 20 min at 65 °C. .. For cleavage assays, FnCas12a (10 μM in SEC buffer) was mixed with crRNA1 (10 μM in H2 O) in the presence of 7.1 mM MgCl2 and incubated at 37 °C for 10 minutes to allow binary complex formation, followed by the additional of linearized plasmid.

    Article Title: Subtle changes to polymer structure and degradation mechanism enable highly effective nanoparticles for siRNA and DNA delivery to human brain cancer
    Article Snippet: For a smaller sample, pUC19 was digested with sspI-HF and pvuII restriction enzymes (New England Biolabs), yielding four fragments (322, 491, and 1873 bp). .. This was then ligated using T4 DNA ligase (New England Biolabs) at room temperature for 2 hr, transformed into DH5α competent cells (Invitrogen), plated onto LB Agar (Sigma) with 100 mg/mL carbenicillin, and incubated for 24 hr at 37°C.

    Article Title: Downstream promoter interactions of TFIID TAFs facilitate transcription reinitiation
    Article Snippet: Transcription activator Gal4-vp16 (800 ng) was incubated with templates for 5 min at room temperature. .. Downstream DNA and bound proteins were released with 120 U of SspI-HF or PstI-HF digestion (New England Biolabs) in 160 µL of 1× transcription buffer for 30 min at room temperature.

    Article Title: In vitro analysis of RNA polymerase II elongation complex dynamics
    Article Snippet: After 30 min of incubation at 25°C to form PICs, transcription was initiated by adding 2.4 µL of NTP mix (final concentration of 400 µM each ATP, CTP, and UTP and 40 µM 3′-Ome-GTP) to the mixture followed by an incubation at 25°C with occasional gentle mixing every 5 min to keep beads suspended. .. To analyze proteins bound to specific regions on the DNA template, complexes were eluted for 30 min at 25° with rotation in 40 µL of the transcription buffer with 60 U of either SspI-HF or PstI-HF (New England Biolabs) as indicated (see ).

    Mass Spectrometry:

    Article Title: Downstream promoter interactions of TFIID TAFs facilitate transcription reinitiation
    Article Snippet: Paragraph title: Immobilized template binding and quantitative mass spectrometry ... Downstream DNA and bound proteins were released with 120 U of SspI-HF or PstI-HF digestion (New England Biolabs) in 160 µL of 1× transcription buffer for 30 min at room temperature.

    Western Blot:

    Article Title: Fallacy of the Unique Genome: Sequence Diversity within Single Helicobacter pylori Strains
    Article Snippet: Genomic DNA was digested with SspI-HF (New England Biolabs) for 2 h. Digested DNA was separated on a 0.5% agarose gel overnight at 0.75 V/cm and transferred to a nylon membrane. .. For Western blot analysis, bacterial lysates of PMSS1 single-colony isolates containing 4, 2, or 1 copies of cagA were prepared by growth in liquid culture to mid-exponential phase followed by sonication on ice.

    Transformation Assay:

    Article Title: Subtle changes to polymer structure and degradation mechanism enable highly effective nanoparticles for siRNA and DNA delivery to human brain cancer
    Article Snippet: For a smaller sample, pUC19 was digested with sspI-HF and pvuII restriction enzymes (New England Biolabs), yielding four fragments (322, 491, and 1873 bp). .. This was then ligated using T4 DNA ligase (New England Biolabs) at room temperature for 2 hr, transformed into DH5α competent cells (Invitrogen), plated onto LB Agar (Sigma) with 100 mg/mL carbenicillin, and incubated for 24 hr at 37°C.

    Hybridization:

    Article Title: Fallacy of the Unique Genome: Sequence Diversity within Single Helicobacter pylori Strains
    Article Snippet: Genomic DNA was digested with SspI-HF (New England Biolabs) for 2 h. Digested DNA was separated on a 0.5% agarose gel overnight at 0.75 V/cm and transferred to a nylon membrane. .. Hybridization and detection were carried out with a North2South chemiluminescent hybridization and detection kit according to the manufacturer’s instructions. cagA copy numbers were determined based on fragment size and the known restriction map of the cagA locus ( ).

    Article Title: The HIV Protease Inhibitor Nelfinavir Inhibits Kaposi's Sarcoma-Associated Herpesvirus Replication In Vitro ▿
    Article Snippet: Twenty micrograms of each DNA sample was digested overnight at 37°C with either AflII-HF or SspI-HF restriction endonucleases according to the manufacturer's recommendations (New England BioLabs, Ipswich, MA). .. Gel electrophoresis, alkaline transfer, Southern blot hybridization, and stringency washes were performed as described by Sambrook and Russell ( ).

    Southern Blot:

    Article Title: Fallacy of the Unique Genome: Sequence Diversity within Single Helicobacter pylori Strains
    Article Snippet: To determine the copy number of cagA in the SS1 and PMSS1 genomes, a Southern blot was performed. .. Genomic DNA was digested with SspI-HF (New England Biolabs) for 2 h. Digested DNA was separated on a 0.5% agarose gel overnight at 0.75 V/cm and transferred to a nylon membrane.

    Article Title: The HIV Protease Inhibitor Nelfinavir Inhibits Kaposi's Sarcoma-Associated Herpesvirus Replication In Vitro ▿
    Article Snippet: Twenty micrograms of each DNA sample was digested overnight at 37°C with either AflII-HF or SspI-HF restriction endonucleases according to the manufacturer's recommendations (New England BioLabs, Ipswich, MA). .. Gel electrophoresis, alkaline transfer, Southern blot hybridization, and stringency washes were performed as described by Sambrook and Russell ( ).

    Generated:

    Article Title: Structural basis for guide RNA processing and seed-dependent DNA targeting and cleavage by CRISPR-Cas12a
    Article Snippet: Target DNA sequence complementary to crRNA1 was generated by annealing complementary synthetic oligonucleotides ( ) and inserting the productinto pUC19 between at the EcoRI and HindIII restriction sites. .. Plasmids were linearized by incubating 640 ng of plasmid DNA with 15 units of SspI-HF (New England Biolabs) in CutSmart buffer (New England Biolabs) for 1 h at 37 °C, followed by heat-inactivation for 20 min at 65 °C.

    Polymerase Chain Reaction:

    Article Title: Fallacy of the Unique Genome: Sequence Diversity within Single Helicobacter pylori Strains
    Article Snippet: Genomic DNA was digested with SspI-HF (New England Biolabs) for 2 h. Digested DNA was separated on a 0.5% agarose gel overnight at 0.75 V/cm and transferred to a nylon membrane. .. A fragment of cagA was PCR amplified from SS1 (bp 1217 to 1514) with primers D008 and R008 ( ) and labeled with biotin using a North2South biotin Random Prime Labeling kit (Thermo Scientific).

    Article Title: Comparison of diets for Largemouth and Smallmouth Bass in Eastern Lake Ontario using DNA barcoding and stable isotope analysis
    Article Snippet: .. Restriction digest of fish prey PCRs occurred in 12.5 μl reactions consisting of; 5 μl PCR product; 1x CutSmart Buffer; and 1 U SspI-HF (New England Biolabs), incubated for 30 min at 37°C. .. PCR and restriction digest success was evaluated by running total digest volumes in 2% agarose gels.

    Sonication:

    Article Title: Fallacy of the Unique Genome: Sequence Diversity within Single Helicobacter pylori Strains
    Article Snippet: Genomic DNA was digested with SspI-HF (New England Biolabs) for 2 h. Digested DNA was separated on a 0.5% agarose gel overnight at 0.75 V/cm and transferred to a nylon membrane. .. For Western blot analysis, bacterial lysates of PMSS1 single-colony isolates containing 4, 2, or 1 copies of cagA were prepared by growth in liquid culture to mid-exponential phase followed by sonication on ice.

    Recombinant:

    Article Title: The HIV Protease Inhibitor Nelfinavir Inhibits Kaposi's Sarcoma-Associated Herpesvirus Replication In Vitro ▿
    Article Snippet: Total genomic DNA was purified from rKSHV.294 recombinant or JSC-1 wild-type virus-infected cells by standard methods ( ). .. Twenty micrograms of each DNA sample was digested overnight at 37°C with either AflII-HF or SspI-HF restriction endonucleases according to the manufacturer's recommendations (New England BioLabs, Ipswich, MA).

    Article Title: In vitro analysis of RNA polymerase II elongation complex dynamics
    Article Snippet: Recombinant transcription activator Gal4-VP16 (400 ng) was preincubated with immobilized templates for 5 min at 25°C prior to the addition of yeast nuclear extract. .. To analyze proteins bound to specific regions on the DNA template, complexes were eluted for 30 min at 25° with rotation in 40 µL of the transcription buffer with 60 U of either SspI-HF or PstI-HF (New England Biolabs) as indicated (see ).

    Nucleic Acid Electrophoresis:

    Article Title: The HIV Protease Inhibitor Nelfinavir Inhibits Kaposi's Sarcoma-Associated Herpesvirus Replication In Vitro ▿
    Article Snippet: Twenty micrograms of each DNA sample was digested overnight at 37°C with either AflII-HF or SspI-HF restriction endonucleases according to the manufacturer's recommendations (New England BioLabs, Ipswich, MA). .. Gel electrophoresis, alkaline transfer, Southern blot hybridization, and stringency washes were performed as described by Sambrook and Russell ( ).

    Mutagenesis:

    Article Title: Structural basis for guide RNA processing and seed-dependent DNA targeting and cleavage by CRISPR-Cas12a
    Article Snippet: Mutations in the target region were introduced by Quikchange Site Directed Mutagenesis ( ). .. Plasmids were linearized by incubating 640 ng of plasmid DNA with 15 units of SspI-HF (New England Biolabs) in CutSmart buffer (New England Biolabs) for 1 h at 37 °C, followed by heat-inactivation for 20 min at 65 °C.

    Isolation:

    Article Title: Subtle changes to polymer structure and degradation mechanism enable highly effective nanoparticles for siRNA and DNA delivery to human brain cancer
    Article Snippet: In order to test a wider range of sizes and structures, the circular pEGFP-N1 plasmid was digested with BsrGI restriction enzyme (New England Biolabs, Ipswich, MA), yielding a single band corresponding to the linearized 4733 bp plasmid, which was isolated and extracted using a gel extraction kit (Qiagen, Valencia, CA) according to the manufacturer’s protocol. .. For a smaller sample, pUC19 was digested with sspI-HF and pvuII restriction enzymes (New England Biolabs), yielding four fragments (322, 491, and 1873 bp).

    Size-exclusion Chromatography:

    Article Title: Structural basis for guide RNA processing and seed-dependent DNA targeting and cleavage by CRISPR-Cas12a
    Article Snippet: Plasmids were linearized by incubating 640 ng of plasmid DNA with 15 units of SspI-HF (New England Biolabs) in CutSmart buffer (New England Biolabs) for 1 h at 37 °C, followed by heat-inactivation for 20 min at 65 °C. .. For cleavage assays, FnCas12a (10 μM in SEC buffer) was mixed with crRNA1 (10 μM in H2 O) in the presence of 7.1 mM MgCl2 and incubated at 37 °C for 10 minutes to allow binary complex formation, followed by the additional of linearized plasmid.

    Labeling:

    Article Title: Fallacy of the Unique Genome: Sequence Diversity within Single Helicobacter pylori Strains
    Article Snippet: Genomic DNA was digested with SspI-HF (New England Biolabs) for 2 h. Digested DNA was separated on a 0.5% agarose gel overnight at 0.75 V/cm and transferred to a nylon membrane. .. A fragment of cagA was PCR amplified from SS1 (bp 1217 to 1514) with primers D008 and R008 ( ) and labeled with biotin using a North2South biotin Random Prime Labeling kit (Thermo Scientific).

    Purification:

    Article Title: The HIV Protease Inhibitor Nelfinavir Inhibits Kaposi's Sarcoma-Associated Herpesvirus Replication In Vitro ▿
    Article Snippet: Total genomic DNA was purified from rKSHV.294 recombinant or JSC-1 wild-type virus-infected cells by standard methods ( ). .. Twenty micrograms of each DNA sample was digested overnight at 37°C with either AflII-HF or SspI-HF restriction endonucleases according to the manufacturer's recommendations (New England BioLabs, Ipswich, MA).

    Article Title: Subtle changes to polymer structure and degradation mechanism enable highly effective nanoparticles for siRNA and DNA delivery to human brain cancer
    Article Snippet: For a smaller sample, pUC19 was digested with sspI-HF and pvuII restriction enzymes (New England Biolabs), yielding four fragments (322, 491, and 1873 bp). .. The 1873-bp fragment containing the ampR gene and origin of replication was isolated and purified as above.

    Sequencing:

    Article Title: Structural basis for guide RNA processing and seed-dependent DNA targeting and cleavage by CRISPR-Cas12a
    Article Snippet: Target DNA sequence complementary to crRNA1 was generated by annealing complementary synthetic oligonucleotides ( ) and inserting the productinto pUC19 between at the EcoRI and HindIII restriction sites. .. Plasmids were linearized by incubating 640 ng of plasmid DNA with 15 units of SspI-HF (New England Biolabs) in CutSmart buffer (New England Biolabs) for 1 h at 37 °C, followed by heat-inactivation for 20 min at 65 °C.

    Staining:

    Article Title: In vitro Enzymology of Cas9
    Article Snippet: For pUC19-based plasmids, use 50 units of SspI-HF (New England Biolabs) and 5 μg plasmid in 1 x CutSmart™ buffer in a total volume of 50 μl. .. Analyze plasmid cleavage by electrophoresis on a 1 % agarose gel in 1 x TAE buffer (40 mM Tris, pH 8.0, 20 mM glacial acetic acid, 1 mM EDTA, pH 8.0) stained with GelRed (Biotium) or similar nucleic acid stain.

    Plasmid Preparation:

    Article Title: In vitro Enzymology of Cas9
    Article Snippet: .. For pUC19-based plasmids, use 50 units of SspI-HF (New England Biolabs) and 5 μg plasmid in 1 x CutSmart™ buffer in a total volume of 50 μl. ..

    Article Title: Structural basis for guide RNA processing and seed-dependent DNA targeting and cleavage by CRISPR-Cas12a
    Article Snippet: .. Plasmids were linearized by incubating 640 ng of plasmid DNA with 15 units of SspI-HF (New England Biolabs) in CutSmart buffer (New England Biolabs) for 1 h at 37 °C, followed by heat-inactivation for 20 min at 65 °C. .. For cleavage assays, FnCas12a (10 μM in SEC buffer) was mixed with crRNA1 (10 μM in H2 O) in the presence of 7.1 mM MgCl2 and incubated at 37 °C for 10 minutes to allow binary complex formation, followed by the additional of linearized plasmid.

    Article Title: Subtle changes to polymer structure and degradation mechanism enable highly effective nanoparticles for siRNA and DNA delivery to human brain cancer
    Article Snippet: In order to test a wider range of sizes and structures, the circular pEGFP-N1 plasmid was digested with BsrGI restriction enzyme (New England Biolabs, Ipswich, MA), yielding a single band corresponding to the linearized 4733 bp plasmid, which was isolated and extracted using a gel extraction kit (Qiagen, Valencia, CA) according to the manufacturer’s protocol. .. For a smaller sample, pUC19 was digested with sspI-HF and pvuII restriction enzymes (New England Biolabs), yielding four fragments (322, 491, and 1873 bp).

    DNA Hybridization:

    Article Title: The HIV Protease Inhibitor Nelfinavir Inhibits Kaposi's Sarcoma-Associated Herpesvirus Replication In Vitro ▿
    Article Snippet: Paragraph title: Viral DNA hybridization. ... Twenty micrograms of each DNA sample was digested overnight at 37°C with either AflII-HF or SspI-HF restriction endonucleases according to the manufacturer's recommendations (New England BioLabs, Ipswich, MA).

    Binding Assay:

    Article Title: Downstream promoter interactions of TFIID TAFs facilitate transcription reinitiation
    Article Snippet: Paragraph title: Immobilized template binding and quantitative mass spectrometry ... Downstream DNA and bound proteins were released with 120 U of SspI-HF or PstI-HF digestion (New England Biolabs) in 160 µL of 1× transcription buffer for 30 min at room temperature.

    Agarose Gel Electrophoresis:

    Article Title: In vitro Enzymology of Cas9
    Article Snippet: For pUC19-based plasmids, use 50 units of SspI-HF (New England Biolabs) and 5 μg plasmid in 1 x CutSmart™ buffer in a total volume of 50 μl. .. Analyze plasmid cleavage by electrophoresis on a 1 % agarose gel in 1 x TAE buffer (40 mM Tris, pH 8.0, 20 mM glacial acetic acid, 1 mM EDTA, pH 8.0) stained with GelRed (Biotium) or similar nucleic acid stain.

    Article Title: Fallacy of the Unique Genome: Sequence Diversity within Single Helicobacter pylori Strains
    Article Snippet: .. Genomic DNA was digested with SspI-HF (New England Biolabs) for 2 h. Digested DNA was separated on a 0.5% agarose gel overnight at 0.75 V/cm and transferred to a nylon membrane. .. A fragment of cagA was PCR amplified from SS1 (bp 1217 to 1514) with primers D008 and R008 ( ) and labeled with biotin using a North2South biotin Random Prime Labeling kit (Thermo Scientific).

    Quantitation Assay:

    Article Title: Downstream promoter interactions of TFIID TAFs facilitate transcription reinitiation
    Article Snippet: Downstream DNA and bound proteins were released with 120 U of SspI-HF or PstI-HF digestion (New England Biolabs) in 160 µL of 1× transcription buffer for 30 min at room temperature. .. For relative quantitation, iTRAQ reporter signal intensity values of all unique peptide scans for a given protein were summed prior to calculation of ratios.

    Sampling:

    Article Title: Comparison of diets for Largemouth and Smallmouth Bass in Eastern Lake Ontario using DNA barcoding and stable isotope analysis
    Article Snippet: Round Goby RFLP identification COI barcode vouchers (n = 2708) from the 112 fish species endemic to the sampling region [ ] were obtained from Barcode of Life Data Systems (BOLD) [ ] and analyzed in R using a script written for this study (see Data Accessibility). .. Restriction digest of fish prey PCRs occurred in 12.5 μl reactions consisting of; 5 μl PCR product; 1x CutSmart Buffer; and 1 U SspI-HF (New England Biolabs), incubated for 30 min at 37°C.

    Concentration Assay:

    Article Title: The HIV Protease Inhibitor Nelfinavir Inhibits Kaposi's Sarcoma-Associated Herpesvirus Replication In Vitro ▿
    Article Snippet: Twenty micrograms of each DNA sample was digested overnight at 37°C with either AflII-HF or SspI-HF restriction endonucleases according to the manufacturer's recommendations (New England BioLabs, Ipswich, MA). .. To generate biotin-labeled probes, PCRs were carried out as described above except that the starting concentration of dTTP was reduced from 200 to 120 μM, and reaction mixtures were supplemented with 80 μM biotin-16-dUTP (Biotium, Hayward, CA).

    Article Title: In vitro analysis of RNA polymerase II elongation complex dynamics
    Article Snippet: After 30 min of incubation at 25°C to form PICs, transcription was initiated by adding 2.4 µL of NTP mix (final concentration of 400 µM each ATP, CTP, and UTP and 40 µM 3′-Ome-GTP) to the mixture followed by an incubation at 25°C with occasional gentle mixing every 5 min to keep beads suspended. .. To analyze proteins bound to specific regions on the DNA template, complexes were eluted for 30 min at 25° with rotation in 40 µL of the transcription buffer with 60 U of either SspI-HF or PstI-HF (New England Biolabs) as indicated (see ).

    Gel Extraction:

    Article Title: Subtle changes to polymer structure and degradation mechanism enable highly effective nanoparticles for siRNA and DNA delivery to human brain cancer
    Article Snippet: In order to test a wider range of sizes and structures, the circular pEGFP-N1 plasmid was digested with BsrGI restriction enzyme (New England Biolabs, Ipswich, MA), yielding a single band corresponding to the linearized 4733 bp plasmid, which was isolated and extracted using a gel extraction kit (Qiagen, Valencia, CA) according to the manufacturer’s protocol. .. For a smaller sample, pUC19 was digested with sspI-HF and pvuII restriction enzymes (New England Biolabs), yielding four fragments (322, 491, and 1873 bp).

    Fluorescence In Situ Hybridization:

    Article Title: Comparison of diets for Largemouth and Smallmouth Bass in Eastern Lake Ontario using DNA barcoding and stable isotope analysis
    Article Snippet: .. Restriction digest of fish prey PCRs occurred in 12.5 μl reactions consisting of; 5 μl PCR product; 1x CutSmart Buffer; and 1 U SspI-HF (New England Biolabs), incubated for 30 min at 37°C. .. PCR and restriction digest success was evaluated by running total digest volumes in 2% agarose gels.

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  • 90
    New England Biolabs sspi hf
    H. pylori SS1 and PMSS1 are gene copy number variable at the cagA locus. (A) Schematic diagram showing tandem arrays of the identical 5,072-bp repeat regions at the cagA locus, all of which contain identical copies of the 3,540-bp cagA gene. The <t>SspI</t> fragment sizes of strains with 1 to 4 cagA copies are shown at the right. The drawing is not to scale. (B) Southern blot of SspI-digested genomic <t>DNA</t> from H. pylori SS1 or PMSS1 probed with a 297-bp PCR product amplified from SS1 cagA bp 1217 to 1514. The original working stocks of SS1 (lane 1) and PMSS1 (lane 5) showed bands corresponding in size to 4, 3, 2, and 1 copies of cagA (asterisks). Subculture of 4 single colonies from the freezer stocks demonstrated clones with 4, 3, or 2 copies of cagA (lanes 2 to 4) from SS1; subculture of 12 single colonies showed either 4 or 2 copies of cagA in PMSS1 (lanes 6 to 8). PMSS1 with a cagA deletion served as a negative control (lane 9). A kilobase ladder is shown at the left. (C) Western blot of H. pylori PMSS1 to examine whether the cagA copy number is positively correlated with protein expression. For this analysis, six individual single-colony isolates of PMSS1 were used, two each with four copies, two copies, or one copy of cagA . Relative quantities of protein in each band were determined using Image Lab software (Bio-Rad Laboratories). The density of the CagA band was divided by the density of the corresponding UreB band to obtain the normalized quantity of CagA to account for differences in gel loading.
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    H. pylori SS1 and PMSS1 are gene copy number variable at the cagA locus. (A) Schematic diagram showing tandem arrays of the identical 5,072-bp repeat regions at the cagA locus, all of which contain identical copies of the 3,540-bp cagA gene. The SspI fragment sizes of strains with 1 to 4 cagA copies are shown at the right. The drawing is not to scale. (B) Southern blot of SspI-digested genomic DNA from H. pylori SS1 or PMSS1 probed with a 297-bp PCR product amplified from SS1 cagA bp 1217 to 1514. The original working stocks of SS1 (lane 1) and PMSS1 (lane 5) showed bands corresponding in size to 4, 3, 2, and 1 copies of cagA (asterisks). Subculture of 4 single colonies from the freezer stocks demonstrated clones with 4, 3, or 2 copies of cagA (lanes 2 to 4) from SS1; subculture of 12 single colonies showed either 4 or 2 copies of cagA in PMSS1 (lanes 6 to 8). PMSS1 with a cagA deletion served as a negative control (lane 9). A kilobase ladder is shown at the left. (C) Western blot of H. pylori PMSS1 to examine whether the cagA copy number is positively correlated with protein expression. For this analysis, six individual single-colony isolates of PMSS1 were used, two each with four copies, two copies, or one copy of cagA . Relative quantities of protein in each band were determined using Image Lab software (Bio-Rad Laboratories). The density of the CagA band was divided by the density of the corresponding UreB band to obtain the normalized quantity of CagA to account for differences in gel loading.

    Journal: mBio

    Article Title: Fallacy of the Unique Genome: Sequence Diversity within Single Helicobacter pylori Strains

    doi: 10.1128/mBio.02321-16

    Figure Lengend Snippet: H. pylori SS1 and PMSS1 are gene copy number variable at the cagA locus. (A) Schematic diagram showing tandem arrays of the identical 5,072-bp repeat regions at the cagA locus, all of which contain identical copies of the 3,540-bp cagA gene. The SspI fragment sizes of strains with 1 to 4 cagA copies are shown at the right. The drawing is not to scale. (B) Southern blot of SspI-digested genomic DNA from H. pylori SS1 or PMSS1 probed with a 297-bp PCR product amplified from SS1 cagA bp 1217 to 1514. The original working stocks of SS1 (lane 1) and PMSS1 (lane 5) showed bands corresponding in size to 4, 3, 2, and 1 copies of cagA (asterisks). Subculture of 4 single colonies from the freezer stocks demonstrated clones with 4, 3, or 2 copies of cagA (lanes 2 to 4) from SS1; subculture of 12 single colonies showed either 4 or 2 copies of cagA in PMSS1 (lanes 6 to 8). PMSS1 with a cagA deletion served as a negative control (lane 9). A kilobase ladder is shown at the left. (C) Western blot of H. pylori PMSS1 to examine whether the cagA copy number is positively correlated with protein expression. For this analysis, six individual single-colony isolates of PMSS1 were used, two each with four copies, two copies, or one copy of cagA . Relative quantities of protein in each band were determined using Image Lab software (Bio-Rad Laboratories). The density of the CagA band was divided by the density of the corresponding UreB band to obtain the normalized quantity of CagA to account for differences in gel loading.

    Article Snippet: Genomic DNA was digested with SspI-HF (New England Biolabs) for 2 h. Digested DNA was separated on a 0.5% agarose gel overnight at 0.75 V/cm and transferred to a nylon membrane.

    Techniques: Southern Blot, Polymerase Chain Reaction, Amplification, Clone Assay, Negative Control, Western Blot, Expressing, Software