restriction enzyme xhoi  (Thermo Fisher)


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  • 99
    Name:
    XhoI
    Description:
    5 C ↓T C G A G 3 3 G A G C T ↑C 5 Thermo Scientific XhoI restriction enzyme recognizes C TCGAG sites and cuts best at 37°C in R buffer See Reaction Conditions for Restriction Enzymes for a table of enzyme activity conditions for double digestion and heat inactivation for this and other restriction enzymes Isoschizomers PaeR7I Sfr274I SlaI StrI TliI Thermo Scientific conventional restriction endonucleases are a large collection of high quality restriction enzymes optimized to work in one of the buffers of the Five Buffer System In addition the universal Tango buffer is provided for convenience in double digestions All of the enzymes exhibit 100 activity in the recommended buffer and reaction conditions To ensure consistent performance Thermo Scientific restriction enzyme reaction buffers contain premixed BSA which enhances the stability of many enzymes and binds contaminants that may be present in DNA preparations Features• Superior quality stringent quality control and industry leading manufacturing process• Convenient color coded Five Buffer System• Includes universal Tango buffer for double digestions• BSA premixed in reaction buffers• Wide selection of restriction endonuclease specificitiesApplications• Molecular cloning• Restriction site mapping• Genotyping• Southern blotting• Restriction fragment length polymorphism RFLP • SNPNote For methylation sensitivity refer to product specifications
    Catalog Number:
    er0693
    Price:
    None
    Applications:
    Cloning|Restriction Enzyme Cloning
    Category:
    Proteins Enzymes Peptides
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    Structured Review

    Thermo Fisher restriction enzyme xhoi
    Southern blot analysis of Δ pksP mutant generated in the Af293 background. (A) Schematic representation of the genomic locus of the Af293 and Δ pksP strains. Deletion of the pksP gene was carried out using the HygR cassette. The cleavage sites of the dual in vitro -assembled Cas9 RNPs are marked by thick vertical lines. <t>XhoI</t> cutting sites are indicated in the pksP locus of the wild-type and Δ pksP strains. (B) Southern blot analysis of 6 arbitrarily selected colonies after digesting genomic <t>DNA</t> with the XhoI restriction enzyme. The wild type (WT) produced a 1.8-kb band that matches the expected wild-type banding pattern. Lanes 1, 2, 4, 5, and 6 displayed a 3.8-kb band which matches the expected pksP deletion banding pattern. The colony in lane 3 displayed a 7.6-kb band, likely containing a tandem integration of the HygR repair template at the pksP locus.
    5 C ↓T C G A G 3 3 G A G C T ↑C 5 Thermo Scientific XhoI restriction enzyme recognizes C TCGAG sites and cuts best at 37°C in R buffer See Reaction Conditions for Restriction Enzymes for a table of enzyme activity conditions for double digestion and heat inactivation for this and other restriction enzymes Isoschizomers PaeR7I Sfr274I SlaI StrI TliI Thermo Scientific conventional restriction endonucleases are a large collection of high quality restriction enzymes optimized to work in one of the buffers of the Five Buffer System In addition the universal Tango buffer is provided for convenience in double digestions All of the enzymes exhibit 100 activity in the recommended buffer and reaction conditions To ensure consistent performance Thermo Scientific restriction enzyme reaction buffers contain premixed BSA which enhances the stability of many enzymes and binds contaminants that may be present in DNA preparations Features• Superior quality stringent quality control and industry leading manufacturing process• Convenient color coded Five Buffer System• Includes universal Tango buffer for double digestions• BSA premixed in reaction buffers• Wide selection of restriction endonuclease specificitiesApplications• Molecular cloning• Restriction site mapping• Genotyping• Southern blotting• Restriction fragment length polymorphism RFLP • SNPNote For methylation sensitivity refer to product specifications
    https://www.bioz.com/result/restriction enzyme xhoi/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    restriction enzyme xhoi - by Bioz Stars, 2020-03
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    Images

    1) Product Images from "A Simple and Universal System for Gene Manipulation in Aspergillus fumigatus: In Vitro-Assembled Cas9-Guide RNA Ribonucleoproteins Coupled with Microhomology Repair Templates"

    Article Title: A Simple and Universal System for Gene Manipulation in Aspergillus fumigatus: In Vitro-Assembled Cas9-Guide RNA Ribonucleoproteins Coupled with Microhomology Repair Templates

    Journal: mSphere

    doi: 10.1128/mSphere.00446-17

    Southern blot analysis of Δ pksP mutant generated in the Af293 background. (A) Schematic representation of the genomic locus of the Af293 and Δ pksP strains. Deletion of the pksP gene was carried out using the HygR cassette. The cleavage sites of the dual in vitro -assembled Cas9 RNPs are marked by thick vertical lines. XhoI cutting sites are indicated in the pksP locus of the wild-type and Δ pksP strains. (B) Southern blot analysis of 6 arbitrarily selected colonies after digesting genomic DNA with the XhoI restriction enzyme. The wild type (WT) produced a 1.8-kb band that matches the expected wild-type banding pattern. Lanes 1, 2, 4, 5, and 6 displayed a 3.8-kb band which matches the expected pksP deletion banding pattern. The colony in lane 3 displayed a 7.6-kb band, likely containing a tandem integration of the HygR repair template at the pksP locus.
    Figure Legend Snippet: Southern blot analysis of Δ pksP mutant generated in the Af293 background. (A) Schematic representation of the genomic locus of the Af293 and Δ pksP strains. Deletion of the pksP gene was carried out using the HygR cassette. The cleavage sites of the dual in vitro -assembled Cas9 RNPs are marked by thick vertical lines. XhoI cutting sites are indicated in the pksP locus of the wild-type and Δ pksP strains. (B) Southern blot analysis of 6 arbitrarily selected colonies after digesting genomic DNA with the XhoI restriction enzyme. The wild type (WT) produced a 1.8-kb band that matches the expected wild-type banding pattern. Lanes 1, 2, 4, 5, and 6 displayed a 3.8-kb band which matches the expected pksP deletion banding pattern. The colony in lane 3 displayed a 7.6-kb band, likely containing a tandem integration of the HygR repair template at the pksP locus.

    Techniques Used: Southern Blot, Mutagenesis, Generated, In Vitro, Produced

    Related Articles

    Clone Assay:

    Article Title: Cloning, Expression, and Purification of Hyperthermophile α-Amylase from Pyrococcus woesei
    Article Snippet: P. woesei chromosomal DNA was used as a source of the α-amylase gene and polymerase chain reaction (PCR) amplification was done with the following primers: 5′- GCTAGC TTGGAGCTTGAAGAGGGAG-3′ and 5′- GAGACC ACAATAACTCCATACGGAG-3′ containing recognition sites for restriction endonucleases Nhe I and Xho I (Fermentas; Vilnius, Lithuania). .. The amplified gene was cloned into pTZ57R/T (InsTAclone PCR Cloning Kit; Fermentas), and then subcloned into pTYB2 and transformed into E. coli BL21 (DE3) pLysS cells.

    Article Title: A secretory hexokinase plays an active role in the proliferation of Nosema bombycis
    Article Snippet: The DsRed, a red fluorescent protein, was cloned used the forward primers 5′-GGAGGCTCAGGGTCAATGGTGCGCTCCTCCAAGAAC-3′ containing a G3 S2 linker sequence (GGAGGCTCAGGGTCA) and the reverse primer 5′-CCTCGAGGCGGCCGCTACAGGAACAGG-3′ containing a Xho I restriction site (CTCGAG). .. The above vectors and pIZ/V5-His (Thermo Fisher, Santa Clara, CA, USA) were digested by Kpn I and Xho I (Thermo Fisher, CA, USA).

    Article Title: Periplasmic Expression of TNF Related Apoptosis Inducing Ligand (TRAIL) in E.coli
    Article Snippet: In the next step, the PCR product was purified and digested with Nde I and Xho I (Fermentas, Lithuania) and then ligated to the corresponding sites of the expression vector pET22b (Novagen, U.S) using T4 DNA ligase (Fermentas, Lithuania) ( ). .. The resulting plasmid was named OmpA-TRAIL/pET22b and the correct cloning was confirmed by PCR and sequencing.

    Article Title: RNA interference targeting virion core protein ORF095 inhibits Goatpox virus replication in Vero cells
    Article Snippet: .. PCR products were digested with Xho I and Bam HI, and cloned into the pEGFP-N1 expression vector (Invitrogen, Inc., Shanghai, China). .. The final construct p095/EGFP (Figure ) was analyzed by restriction digestion and sequenced.

    Article Title: Human endogenous retroviral syncytin exerts inhibitory effect on invasive phenotype of B16F10 melanoma cells
    Article Snippet: Paragraph title: Cloning and sequencing of syncytin ... The plasmids that contained the desired inserts were identified by double restriction enzyme digestion with Xho I and Eco R I, and DNA sequencing performed by Invitrogen (Guangzhou, China).

    Article Title: Exposition of hepatitis B surface antigen (HBsAg) on the surface of HEK293T cell and evaluation of its expression
    Article Snippet: .. The extracted plasmid of one of the resulted clones and pcDNA 3.1 Hygro (+) plasmid (Invitrogen, USA) were digested by NheI and XhoI restriction enzymes (Thermoscience, USA) separately. .. The digestion products were electro-phoresed and purified from agarose gel by Bioneer DNA extraction kit (South Korea).

    Amplification:

    Article Title: Cloning, Expression, and Purification of Hyperthermophile α-Amylase from Pyrococcus woesei
    Article Snippet: .. P. woesei chromosomal DNA was used as a source of the α-amylase gene and polymerase chain reaction (PCR) amplification was done with the following primers: 5′- GCTAGC TTGGAGCTTGAAGAGGGAG-3′ and 5′- GAGACC ACAATAACTCCATACGGAG-3′ containing recognition sites for restriction endonucleases Nhe I and Xho I (Fermentas; Vilnius, Lithuania). .. The reaction was performed using 10 ng DNA, 2 μL (10 μM) of each primer, 5 μL (10mM) dNTPs, 5 μL 10× PCR buffer [100mM Tris-HCl (pH 8.9), 500mM KCl, 20mM MgCl2 , 1% Triton X-100] and High-fidelity PCR Enzyme Mix (Fermentas).

    Article Title: A Simple and Universal System for Gene Manipulation in Aspergillus fumigatus: In Vitro-Assembled Cas9-Guide RNA Ribonucleoproteins Coupled with Microhomology Repair Templates
    Article Snippet: A pksP -specific probe was amplified from genomic DNA of Af293 by using the primer set pksP-Probe-Forward and pksP-Probe-Reverse ( ). .. Following quanti fication by a NanoDrop spectrophotometer and integrity verification by agarose gel electrophoresis, the genomic DNA was digested overnight at 37°C using the restriction enzyme XhoI and was separated on an agarose gel before being transferred to a Biodyne B modified nylon membrane (Thermo Scientific).

    Article Title: RNA interference targeting virion core protein ORF095 inhibits Goatpox virus replication in Vero cells
    Article Snippet: Directed cloning PCR was used to amplify the ORF095 gene, using the following primers (sense: 5'-GTC CTCGAG ATGGACTTCATGAAAAAATATACTAA-3' and antisense: 5'-GC GGATCC TTGCTGTTATTATCATCTAGTTTG-3') used for amplification contained the target sequences for Xho I ( CTCGAG ) and Bam HI ( GGATCC ) incorporated at the 5' of the viral complementary sequence. .. PCR products were digested with Xho I and Bam HI, and cloned into the pEGFP-N1 expression vector (Invitrogen, Inc., Shanghai, China).

    Article Title: Human endogenous retroviral syncytin exerts inhibitory effect on invasive phenotype of B16F10 melanoma cells
    Article Snippet: The following PCR program was used for cDNA amplification: initial denaturation at 94 °C for 2 min, 35 cycles of 94 °C for 30 s, 55 °C for 30 s and 72 °C for 2 min, and a final extension at 72 °C for 10 min. .. The plasmids that contained the desired inserts were identified by double restriction enzyme digestion with Xho I and Eco R I, and DNA sequencing performed by Invitrogen (Guangzhou, China).

    Synthesized:

    Article Title: Development and Application of a Reverse Genetics System for Japanese Encephalitis Virus
    Article Snippet: RNA transcripts were synthesized by in vitro transcription. .. Here, 100 to 200 ng of the template DNA linearized with Xho I or Xba I digestion and in some cases modified with mung bean nuclease was added to a 25-μl reaction mixture consisting of the buffer supplied by the manufacturer (Gibco-BRL) plus 0.6 mM cap analog [m7 G(5′)ppp(5′)A or m7 G(5′)ppp(5′)G; NEB Inc.], 0.5 μM [3 H]UTP (1.0 mCi/ml, 50 Ci/mmol; New England Nuclear Corp., Boston, Mass.), 10 mM DTT, 1 mM each UTP, GTP, CTP, and ATP, 40 U of RNaseOUT, and 15 U of SP6 RNA polymerase (Gibco-BRL).

    Article Title: Exposition of hepatitis B surface antigen (HBsAg) on the surface of HEK293T cell and evaluation of its expression
    Article Snippet: Plasmid construction Coding sequence of HBsAg from Adw subtype of HBV (sequence ID: X02763.1), including start to stop codon, with site of NheI at 5’ end and XhoI at 3’ was ordered to be synthesized and cloned in pUC57 plasmid to Genecust Company (Luxembourg). .. The extracted plasmid of one of the resulted clones and pcDNA 3.1 Hygro (+) plasmid (Invitrogen, USA) were digested by NheI and XhoI restriction enzymes (Thermoscience, USA) separately.

    Construct:

    Article Title: Construction of Eukaryotic Expression Vector with mBD1-mBD3 Fusion Genes and Exploring Its Activity against Influenza A Virus
    Article Snippet: The Construction of Recombinant Plasmid pcDNA3.1(+)/mBD1-mBD3 The overlap-PCR product was cleaved by Eco R I and Xho I (Fermentas Co. Ltd, Shenzhen, China), and the mBD1-mBD3 fragment was inserted into similarly digested pcDNA3.1(+) (Invitrogen, Shanghai, China) vector with T4 DNA ligase (Fermentas Co. Ltd, Shenzhen, China) at 16 °C to construct the expression plasmid named pcDNA3.1(+)/mBD1-mBD3 . .. The inserted sequences were confirmed by PCR, restriction enzyme digestion analysis with Eco R I and Xho I and sequencing using the ABI 377 DNA Analyzer (Invitrogen, Shanghai, China).

    Article Title: A secretory hexokinase plays an active role in the proliferation of Nosema bombycis
    Article Snippet: The above vectors and pIZ/V5-His (Thermo Fisher, Santa Clara, CA, USA) were digested by Kpn I and Xho I (Thermo Fisher, CA, USA). .. Two µg constructed vectors were transiently transfected into BmN cells using X-tremeGENE™ HP DNA Transfection Reagent (Thermo Fisher, Santa Clara, CA, USA).

    Article Title: RNA interference targeting virion core protein ORF095 inhibits Goatpox virus replication in Vero cells
    Article Snippet: PCR products were digested with Xho I and Bam HI, and cloned into the pEGFP-N1 expression vector (Invitrogen, Inc., Shanghai, China). .. The final construct p095/EGFP (Figure ) was analyzed by restriction digestion and sequenced.

    Adsorption:

    Article Title: Development and Application of a Reverse Genetics System for Japanese Encephalitis Virus
    Article Snippet: Here, 100 to 200 ng of the template DNA linearized with Xho I or Xba I digestion and in some cases modified with mung bean nuclease was added to a 25-μl reaction mixture consisting of the buffer supplied by the manufacturer (Gibco-BRL) plus 0.6 mM cap analog [m7 G(5′)ppp(5′)A or m7 G(5′)ppp(5′)G; NEB Inc.], 0.5 μM [3 H]UTP (1.0 mCi/ml, 50 Ci/mmol; New England Nuclear Corp., Boston, Mass.), 10 mM DTT, 1 mM each UTP, GTP, CTP, and ATP, 40 U of RNaseOUT, and 15 U of SP6 RNA polymerase (Gibco-BRL). .. The reaction mixtures were incubated at 37°C for 1 h. RNAs were quantified on the basis of [3 H]UTP incorporation as measured by RNA adsorption to DE-81 (Whatman, Maidstone, United Kingdom) filter paper ( ).

    Electrophoresis:

    Article Title: Bacterial Contig Map of the 21q11 Region Associated with Alzheimer's Disease and Abnormal Myelopoiesis in Down Syndrome
    Article Snippet: PAC DNA (0.50–1 μg) was digested at 37°C with one or a combination of the following restriction enzymes, Not I, Sal I (NEB), Xho I (Life Technologies). .. Electrophoresis conditions were as follows: For the 4- to 200-kb window, 5.2 V/cm, 14 hr, switch time 3–15 sec; for the 100- to 1600-kb window, 6 V/cm, 22 hr, switch time 40–80 sec. DNA size markers used (see Fig. , from left to right) were Lambda ladder (Bio-Rad), high molecular weight DNA markers (Life Technologies), 1-kb DNA ladder (Life Technologies).

    Incubation:

    Article Title: Development and Application of a Reverse Genetics System for Japanese Encephalitis Virus
    Article Snippet: Here, 100 to 200 ng of the template DNA linearized with Xho I or Xba I digestion and in some cases modified with mung bean nuclease was added to a 25-μl reaction mixture consisting of the buffer supplied by the manufacturer (Gibco-BRL) plus 0.6 mM cap analog [m7 G(5′)ppp(5′)A or m7 G(5′)ppp(5′)G; NEB Inc.], 0.5 μM [3 H]UTP (1.0 mCi/ml, 50 Ci/mmol; New England Nuclear Corp., Boston, Mass.), 10 mM DTT, 1 mM each UTP, GTP, CTP, and ATP, 40 U of RNaseOUT, and 15 U of SP6 RNA polymerase (Gibco-BRL). .. The reaction mixtures were incubated at 37°C for 1 h. RNAs were quantified on the basis of [3 H]UTP incorporation as measured by RNA adsorption to DE-81 (Whatman, Maidstone, United Kingdom) filter paper ( ).

    Article Title: Human endogenous retroviral syncytin exerts inhibitory effect on invasive phenotype of B16F10 melanoma cells
    Article Snippet: The purified PCR products were ligated into pEGFP-N1 vector after being digested with Xho I and Eco R I. Ligations were transformed into Escherichia coli DH5α and 10 bacterial colonies were picked from per primer pair and incubated with shaking overnight in 5 mL LB medium containing 34 µg/mL kanamycin. .. The plasmids that contained the desired inserts were identified by double restriction enzyme digestion with Xho I and Eco R I, and DNA sequencing performed by Invitrogen (Guangzhou, China).

    Expressing:

    Article Title: Construction of Eukaryotic Expression Vector with mBD1-mBD3 Fusion Genes and Exploring Its Activity against Influenza A Virus
    Article Snippet: The Construction of Recombinant Plasmid pcDNA3.1(+)/mBD1-mBD3 The overlap-PCR product was cleaved by Eco R I and Xho I (Fermentas Co. Ltd, Shenzhen, China), and the mBD1-mBD3 fragment was inserted into similarly digested pcDNA3.1(+) (Invitrogen, Shanghai, China) vector with T4 DNA ligase (Fermentas Co. Ltd, Shenzhen, China) at 16 °C to construct the expression plasmid named pcDNA3.1(+)/mBD1-mBD3 . .. The inserted sequences were confirmed by PCR, restriction enzyme digestion analysis with Eco R I and Xho I and sequencing using the ABI 377 DNA Analyzer (Invitrogen, Shanghai, China).

    Article Title: Cloning, Expression, and Purification of Hyperthermophile α-Amylase from Pyrococcus woesei
    Article Snippet: Paragraph title: Construction of expression plasmid ... P. woesei chromosomal DNA was used as a source of the α-amylase gene and polymerase chain reaction (PCR) amplification was done with the following primers: 5′- GCTAGC TTGGAGCTTGAAGAGGGAG-3′ and 5′- GAGACC ACAATAACTCCATACGGAG-3′ containing recognition sites for restriction endonucleases Nhe I and Xho I (Fermentas; Vilnius, Lithuania).

    Article Title: A secretory hexokinase plays an active role in the proliferation of Nosema bombycis
    Article Snippet: Paragraph title: Expression of recombinant HK fused with DsRed in BmN ... The above vectors and pIZ/V5-His (Thermo Fisher, Santa Clara, CA, USA) were digested by Kpn I and Xho I (Thermo Fisher, CA, USA).

    Article Title: Periplasmic Expression of TNF Related Apoptosis Inducing Ligand (TRAIL) in E.coli
    Article Snippet: .. In the next step, the PCR product was purified and digested with Nde I and Xho I (Fermentas, Lithuania) and then ligated to the corresponding sites of the expression vector pET22b (Novagen, U.S) using T4 DNA ligase (Fermentas, Lithuania) ( ). .. The resulting plasmid was named OmpA-TRAIL/pET22b and the correct cloning was confirmed by PCR and sequencing.

    Article Title: RNA interference targeting virion core protein ORF095 inhibits Goatpox virus replication in Vero cells
    Article Snippet: .. PCR products were digested with Xho I and Bam HI, and cloned into the pEGFP-N1 expression vector (Invitrogen, Inc., Shanghai, China). .. The final construct p095/EGFP (Figure ) was analyzed by restriction digestion and sequenced.

    Cell Fractionation:

    Article Title: Periplasmic Expression of TNF Related Apoptosis Inducing Ligand (TRAIL) in E.coli
    Article Snippet: In the next step, the PCR product was purified and digested with Nde I and Xho I (Fermentas, Lithuania) and then ligated to the corresponding sites of the expression vector pET22b (Novagen, U.S) using T4 DNA ligase (Fermentas, Lithuania) ( ). .. Expression of TRAIL and cell fractionation The recombinant plasmid OmpA-TRAIL/pET22b was transformed into the E.coli BL21 (DE3) by treatment with 0.1 M cold CaCl2 and then heat shock at 42 °C for 1 minute.

    Modification:

    Article Title: A Simple and Universal System for Gene Manipulation in Aspergillus fumigatus: In Vitro-Assembled Cas9-Guide RNA Ribonucleoproteins Coupled with Microhomology Repair Templates
    Article Snippet: .. Following quanti fication by a NanoDrop spectrophotometer and integrity verification by agarose gel electrophoresis, the genomic DNA was digested overnight at 37°C using the restriction enzyme XhoI and was separated on an agarose gel before being transferred to a Biodyne B modified nylon membrane (Thermo Scientific). .. Membrane-bound, digested genomic DNA was hybridized with the biotinylated probe and then labeled with horseradish peroxidase-conjugated streptavidin (Thermo Scientific).

    Article Title: Development and Application of a Reverse Genetics System for Japanese Encephalitis Virus
    Article Snippet: .. Here, 100 to 200 ng of the template DNA linearized with Xho I or Xba I digestion and in some cases modified with mung bean nuclease was added to a 25-μl reaction mixture consisting of the buffer supplied by the manufacturer (Gibco-BRL) plus 0.6 mM cap analog [m7 G(5′)ppp(5′)A or m7 G(5′)ppp(5′)G; NEB Inc.], 0.5 μM [3 H]UTP (1.0 mCi/ml, 50 Ci/mmol; New England Nuclear Corp., Boston, Mass.), 10 mM DTT, 1 mM each UTP, GTP, CTP, and ATP, 40 U of RNaseOUT, and 15 U of SP6 RNA polymerase (Gibco-BRL). .. The reaction mixtures were incubated at 37°C for 1 h. RNAs were quantified on the basis of [3 H]UTP incorporation as measured by RNA adsorption to DE-81 (Whatman, Maidstone, United Kingdom) filter paper ( ).

    Transformation Assay:

    Article Title: Cloning, Expression, and Purification of Hyperthermophile α-Amylase from Pyrococcus woesei
    Article Snippet: P. woesei chromosomal DNA was used as a source of the α-amylase gene and polymerase chain reaction (PCR) amplification was done with the following primers: 5′- GCTAGC TTGGAGCTTGAAGAGGGAG-3′ and 5′- GAGACC ACAATAACTCCATACGGAG-3′ containing recognition sites for restriction endonucleases Nhe I and Xho I (Fermentas; Vilnius, Lithuania). .. The amplified gene was cloned into pTZ57R/T (InsTAclone PCR Cloning Kit; Fermentas), and then subcloned into pTYB2 and transformed into E. coli BL21 (DE3) pLysS cells.

    Article Title: A Simple and Universal System for Gene Manipulation in Aspergillus fumigatus: In Vitro-Assembled Cas9-Guide RNA Ribonucleoproteins Coupled with Microhomology Repair Templates
    Article Snippet: The genomic DNA of 6 arbitrarily selected white colonies, which were transformed with 2 µg of the 35-bp-flanked HygR repair template, was isolated using a standard phenol-chloroform extraction protocol. .. Following quanti fication by a NanoDrop spectrophotometer and integrity verification by agarose gel electrophoresis, the genomic DNA was digested overnight at 37°C using the restriction enzyme XhoI and was separated on an agarose gel before being transferred to a Biodyne B modified nylon membrane (Thermo Scientific).

    Article Title: Periplasmic Expression of TNF Related Apoptosis Inducing Ligand (TRAIL) in E.coli
    Article Snippet: In the next step, the PCR product was purified and digested with Nde I and Xho I (Fermentas, Lithuania) and then ligated to the corresponding sites of the expression vector pET22b (Novagen, U.S) using T4 DNA ligase (Fermentas, Lithuania) ( ). .. Expression of TRAIL and cell fractionation The recombinant plasmid OmpA-TRAIL/pET22b was transformed into the E.coli BL21 (DE3) by treatment with 0.1 M cold CaCl2 and then heat shock at 42 °C for 1 minute.

    Article Title: Human endogenous retroviral syncytin exerts inhibitory effect on invasive phenotype of B16F10 melanoma cells
    Article Snippet: The purified PCR products were ligated into pEGFP-N1 vector after being digested with Xho I and Eco R I. Ligations were transformed into Escherichia coli DH5α and 10 bacterial colonies were picked from per primer pair and incubated with shaking overnight in 5 mL LB medium containing 34 µg/mL kanamycin. .. The plasmids that contained the desired inserts were identified by double restriction enzyme digestion with Xho I and Eco R I, and DNA sequencing performed by Invitrogen (Guangzhou, China).

    Article Title: Exposition of hepatitis B surface antigen (HBsAg) on the surface of HEK293T cell and evaluation of its expression
    Article Snippet: This purchased plasmid was transformed to chemically competent E. coli Top 10F’ according to Higa and Mendel protocol. .. The extracted plasmid of one of the resulted clones and pcDNA 3.1 Hygro (+) plasmid (Invitrogen, USA) were digested by NheI and XhoI restriction enzymes (Thermoscience, USA) separately.

    Article Title: Expression of Pennisetum glaucum Eukaryotic Translational Initiation Factor 4A (PgeIF4A) Confers Improved Drought, Salinity, and Oxidative Stress Tolerance in Groundnut
    Article Snippet: .. Copy number detection by southern blotting Genomic DNA (20 μg) from control and transformed plants (T0 ) was digested using 30 U of Xho I (Fermentas life sciences), single cutter of T-DNA and non-cutter of PgeIF4A gene. .. The digested samples were size fractionated on agarose gel (0.8%) for 16 h at 30 V. The depurinated and denatured DNA fragments were subsequently transferred to positively charged Hybond N+ nylon membrane (GE Health Care, USA).

    Hybridization:

    Article Title: A Simple and Universal System for Gene Manipulation in Aspergillus fumigatus: In Vitro-Assembled Cas9-Guide RNA Ribonucleoproteins Coupled with Microhomology Repair Templates
    Article Snippet: Following quanti fication by a NanoDrop spectrophotometer and integrity verification by agarose gel electrophoresis, the genomic DNA was digested overnight at 37°C using the restriction enzyme XhoI and was separated on an agarose gel before being transferred to a Biodyne B modified nylon membrane (Thermo Scientific). .. Finally, the membrane was visualized by using the North2South chemiluminescent hybridization and detection kit (Thermo Scientific).

    Article Title: Plant-derived SAC domain of PAR-4 (Prostate Apoptosis Response 4) exhibits growth inhibitory effects in prostate cancer cells
    Article Snippet: Southern Blotting Southern hybridization was carried out according to . .. Briefly, 10 μg of gDNA isolated from four transgenic (L2, L3, L5, and L6 of SAC-Par-4-GFP) and vector control lines were digested overnight with Xho I (Fermentas) at 37°C, subsequently electrophoresed on 0.8% TAE-agarose and transferred to a nylon membrane (Hybond-N+, Amersham).

    Transfection:

    Article Title: A secretory hexokinase plays an active role in the proliferation of Nosema bombycis
    Article Snippet: The above vectors and pIZ/V5-His (Thermo Fisher, Santa Clara, CA, USA) were digested by Kpn I and Xho I (Thermo Fisher, CA, USA). .. Two µg constructed vectors were transiently transfected into BmN cells using X-tremeGENE™ HP DNA Transfection Reagent (Thermo Fisher, Santa Clara, CA, USA).

    Article Title: RNA interference targeting virion core protein ORF095 inhibits Goatpox virus replication in Vero cells
    Article Snippet: PCR products were digested with Xho I and Bam HI, and cloned into the pEGFP-N1 expression vector (Invitrogen, Inc., Shanghai, China). .. Plasmids used for transfection were purified with the Wizard Purefection TM Plasmid DNA Purification System (Promega, USA) and quantified by Biophotometer (Eppendorf, Germany).

    Article Title: Development and Application of a Reverse Genetics System for Japanese Encephalitis Virus
    Article Snippet: Paragraph title: Transcriptions and transfections. ... Here, 100 to 200 ng of the template DNA linearized with Xho I or Xba I digestion and in some cases modified with mung bean nuclease was added to a 25-μl reaction mixture consisting of the buffer supplied by the manufacturer (Gibco-BRL) plus 0.6 mM cap analog [m7 G(5′)ppp(5′)A or m7 G(5′)ppp(5′)G; NEB Inc.], 0.5 μM [3 H]UTP (1.0 mCi/ml, 50 Ci/mmol; New England Nuclear Corp., Boston, Mass.), 10 mM DTT, 1 mM each UTP, GTP, CTP, and ATP, 40 U of RNaseOUT, and 15 U of SP6 RNA polymerase (Gibco-BRL).

    Southern Blot:

    Article Title: A Simple and Universal System for Gene Manipulation in Aspergillus fumigatus: In Vitro-Assembled Cas9-Guide RNA Ribonucleoproteins Coupled with Microhomology Repair Templates
    Article Snippet: Paragraph title: Southern blot analysis of ΔpksP mutants. ... Following quanti fication by a NanoDrop spectrophotometer and integrity verification by agarose gel electrophoresis, the genomic DNA was digested overnight at 37°C using the restriction enzyme XhoI and was separated on an agarose gel before being transferred to a Biodyne B modified nylon membrane (Thermo Scientific).

    Article Title: Plant-derived SAC domain of PAR-4 (Prostate Apoptosis Response 4) exhibits growth inhibitory effects in prostate cancer cells
    Article Snippet: Paragraph title: Southern Blotting ... Briefly, 10 μg of gDNA isolated from four transgenic (L2, L3, L5, and L6 of SAC-Par-4-GFP) and vector control lines were digested overnight with Xho I (Fermentas) at 37°C, subsequently electrophoresed on 0.8% TAE-agarose and transferred to a nylon membrane (Hybond-N+, Amersham).

    Article Title: Expression of Pennisetum glaucum Eukaryotic Translational Initiation Factor 4A (PgeIF4A) Confers Improved Drought, Salinity, and Oxidative Stress Tolerance in Groundnut
    Article Snippet: .. Copy number detection by southern blotting Genomic DNA (20 μg) from control and transformed plants (T0 ) was digested using 30 U of Xho I (Fermentas life sciences), single cutter of T-DNA and non-cutter of PgeIF4A gene. .. The digested samples were size fractionated on agarose gel (0.8%) for 16 h at 30 V. The depurinated and denatured DNA fragments were subsequently transferred to positively charged Hybond N+ nylon membrane (GE Health Care, USA).

    Ligation:

    Article Title: Exposition of hepatitis B surface antigen (HBsAg) on the surface of HEK293T cell and evaluation of its expression
    Article Snippet: The extracted plasmid of one of the resulted clones and pcDNA 3.1 Hygro (+) plasmid (Invitrogen, USA) were digested by NheI and XhoI restriction enzymes (Thermoscience, USA) separately. .. Ligation reaction was done between linear pcDNA 3.1 Hygro (+) plasmid and HBsAg coding fragments with T4 DNA ligase.

    Overlap Extension Polymerase Chain Reaction:

    Article Title: Periplasmic Expression of TNF Related Apoptosis Inducing Ligand (TRAIL) in E.coli
    Article Snippet: Two forward primers and one reverse primer (MWG, Germany) applied for OE-PCR was shown as . .. In the next step, the PCR product was purified and digested with Nde I and Xho I (Fermentas, Lithuania) and then ligated to the corresponding sites of the expression vector pET22b (Novagen, U.S) using T4 DNA ligase (Fermentas, Lithuania) ( ).

    DNA Sequencing:

    Article Title: Human endogenous retroviral syncytin exerts inhibitory effect on invasive phenotype of B16F10 melanoma cells
    Article Snippet: .. The plasmids that contained the desired inserts were identified by double restriction enzyme digestion with Xho I and Eco R I, and DNA sequencing performed by Invitrogen (Guangzhou, China). .. The plasmid encoding the syncytin-EGFP fusion protein and pEGFP-N1 vector were transfected into B16F10 cells by Trans-EZ according to the manufacturer’s protocol in a 24-well plate, respectively.

    DNA Labeling:

    Article Title: Expression of Pennisetum glaucum Eukaryotic Translational Initiation Factor 4A (PgeIF4A) Confers Improved Drought, Salinity, and Oxidative Stress Tolerance in Groundnut
    Article Snippet: Copy number detection by southern blotting Genomic DNA (20 μg) from control and transformed plants (T0 ) was digested using 30 U of Xho I (Fermentas life sciences), single cutter of T-DNA and non-cutter of PgeIF4A gene. .. Pre-Hybridization, washing and detection of transgene signals were carried out by following manufacturer's instructions (Biotin DNA labeling and chromogenic detection kit-Thermo Scientifics, USA).

    Sequencing:

    Article Title: Construction of Eukaryotic Expression Vector with mBD1-mBD3 Fusion Genes and Exploring Its Activity against Influenza A Virus
    Article Snippet: .. The inserted sequences were confirmed by PCR, restriction enzyme digestion analysis with Eco R I and Xho I and sequencing using the ABI 377 DNA Analyzer (Invitrogen, Shanghai, China). .. Screening of Stable Expression MDCK Stains Transfected with pcDNA3.1(+)/mBD1-mBD3 The final concentration of MDCK cells were adjusted to 2 × 105 –5 × 105 /mL with DMEM (Gibco, Grand Island, NY, USA) supplemented with 10% bovine serum, 100 U/mL penicillin and 100 µg/mL streptomycin.

    Article Title: Loss of prostasin (PRSS8) in human bladder transitional cell carcinoma cell lines is associated with epithelial-mesenchymal transition (EMT)
    Article Snippet: For each cell line, aliquots of 2 μg of DNA were separately digested with 5 units of Xho I ( X ), Hpa II ( H ), or Msp I ( M ) (Invitrogen) in a total volume of 20 μl at 37°C for overnight. .. The upstream primer ends with part of the Hpa II/Msp I sequence at the -96 CpG: CCG G (underlined).

    Article Title: A secretory hexokinase plays an active role in the proliferation of Nosema bombycis
    Article Snippet: The DsRed, a red fluorescent protein, was cloned used the forward primers 5′-GGAGGCTCAGGGTCAATGGTGCGCTCCTCCAAGAAC-3′ containing a G3 S2 linker sequence (GGAGGCTCAGGGTCA) and the reverse primer 5′-CCTCGAGGCGGCCGCTACAGGAACAGG-3′ containing a Xho I restriction site (CTCGAG). .. The above vectors and pIZ/V5-His (Thermo Fisher, Santa Clara, CA, USA) were digested by Kpn I and Xho I (Thermo Fisher, CA, USA).

    Article Title: Periplasmic Expression of TNF Related Apoptosis Inducing Ligand (TRAIL) in E.coli
    Article Snippet: In the next step, the PCR product was purified and digested with Nde I and Xho I (Fermentas, Lithuania) and then ligated to the corresponding sites of the expression vector pET22b (Novagen, U.S) using T4 DNA ligase (Fermentas, Lithuania) ( ). .. The resulting plasmid was named OmpA-TRAIL/pET22b and the correct cloning was confirmed by PCR and sequencing.

    Article Title: RNA interference targeting virion core protein ORF095 inhibits Goatpox virus replication in Vero cells
    Article Snippet: The reverse primer uncontained a TTA sequence, which was used in the characterization of ORF095 protein and EGFP co-expression. .. PCR products were digested with Xho I and Bam HI, and cloned into the pEGFP-N1 expression vector (Invitrogen, Inc., Shanghai, China).

    Article Title: Human endogenous retroviral syncytin exerts inhibitory effect on invasive phenotype of B16F10 melanoma cells
    Article Snippet: Paragraph title: Cloning and sequencing of syncytin ... The plasmids that contained the desired inserts were identified by double restriction enzyme digestion with Xho I and Eco R I, and DNA sequencing performed by Invitrogen (Guangzhou, China).

    Article Title: Exposition of hepatitis B surface antigen (HBsAg) on the surface of HEK293T cell and evaluation of its expression
    Article Snippet: Plasmid construction Coding sequence of HBsAg from Adw subtype of HBV (sequence ID: X02763.1), including start to stop codon, with site of NheI at 5’ end and XhoI at 3’ was ordered to be synthesized and cloned in pUC57 plasmid to Genecust Company (Luxembourg). .. The extracted plasmid of one of the resulted clones and pcDNA 3.1 Hygro (+) plasmid (Invitrogen, USA) were digested by NheI and XhoI restriction enzymes (Thermoscience, USA) separately.

    CpG Methylation Assay:

    Article Title: Loss of prostasin (PRSS8) in human bladder transitional cell carcinoma cell lines is associated with epithelial-mesenchymal transition (EMT)
    Article Snippet: Paragraph title: Analysis of prostasin promoter CpG methylation state by methylation-specific PCR (MSP) ... For each cell line, aliquots of 2 μg of DNA were separately digested with 5 units of Xho I ( X ), Hpa II ( H ), or Msp I ( M ) (Invitrogen) in a total volume of 20 μl at 37°C for overnight.

    Recombinant:

    Article Title: Construction of Eukaryotic Expression Vector with mBD1-mBD3 Fusion Genes and Exploring Its Activity against Influenza A Virus
    Article Snippet: Paragraph title: 2.2. The Construction of Recombinant Plasmid pcDNA3.1(+)/mBD1-mBD3 ... The inserted sequences were confirmed by PCR, restriction enzyme digestion analysis with Eco R I and Xho I and sequencing using the ABI 377 DNA Analyzer (Invitrogen, Shanghai, China).

    Article Title: A secretory hexokinase plays an active role in the proliferation of Nosema bombycis
    Article Snippet: Paragraph title: Expression of recombinant HK fused with DsRed in BmN ... The above vectors and pIZ/V5-His (Thermo Fisher, Santa Clara, CA, USA) were digested by Kpn I and Xho I (Thermo Fisher, CA, USA).

    Article Title: Pluripotent State Induction in Mouse Embryonic Fibroblast Using mRNAs of Reprogramming Factors
    Article Snippet: .. In Vitro Transcription of mRNA To synthesize the mRNA of the inserted transcription factors (TFs), the designed recombinant plasmids were linearized by Xho I, followed by in vitro RNA transcription (IVT) using the mMESSAGE mMACHINE T7 kit (AM1345, Ambion® , Grand Island, NY, USA) that allows 5' cap and poly (A) tail formation. .. The transcribed mRNA was then purified using MEGAclear™ Kit (AM1908, Ambion® , Grand Island, NY, USA) according to the protocol of the manufacturer.

    Article Title: Periplasmic Expression of TNF Related Apoptosis Inducing Ligand (TRAIL) in E.coli
    Article Snippet: In the next step, the PCR product was purified and digested with Nde I and Xho I (Fermentas, Lithuania) and then ligated to the corresponding sites of the expression vector pET22b (Novagen, U.S) using T4 DNA ligase (Fermentas, Lithuania) ( ). .. Expression of TRAIL and cell fractionation The recombinant plasmid OmpA-TRAIL/pET22b was transformed into the E.coli BL21 (DE3) by treatment with 0.1 M cold CaCl2 and then heat shock at 42 °C for 1 minute.

    Molecular Weight:

    Article Title: Loss of prostasin (PRSS8) in human bladder transitional cell carcinoma cell lines is associated with epithelial-mesenchymal transition (EMT)
    Article Snippet: Analysis of prostasin promoter CpG methylation state by methylation-specific PCR (MSP) High molecular weight genomic DNA was extracted from the urothelial cells as described previously [ ]. .. For each cell line, aliquots of 2 μg of DNA were separately digested with 5 units of Xho I ( X ), Hpa II ( H ), or Msp I ( M ) (Invitrogen) in a total volume of 20 μl at 37°C for overnight.

    Article Title: Bacterial Contig Map of the 21q11 Region Associated with Alzheimer's Disease and Abnormal Myelopoiesis in Down Syndrome
    Article Snippet: PAC DNA (0.50–1 μg) was digested at 37°C with one or a combination of the following restriction enzymes, Not I, Sal I (NEB), Xho I (Life Technologies). .. Electrophoresis conditions were as follows: For the 4- to 200-kb window, 5.2 V/cm, 14 hr, switch time 3–15 sec; for the 100- to 1600-kb window, 6 V/cm, 22 hr, switch time 40–80 sec. DNA size markers used (see Fig. , from left to right) were Lambda ladder (Bio-Rad), high molecular weight DNA markers (Life Technologies), 1-kb DNA ladder (Life Technologies).

    DNA Extraction:

    Article Title: Exposition of hepatitis B surface antigen (HBsAg) on the surface of HEK293T cell and evaluation of its expression
    Article Snippet: The extracted plasmid of one of the resulted clones and pcDNA 3.1 Hygro (+) plasmid (Invitrogen, USA) were digested by NheI and XhoI restriction enzymes (Thermoscience, USA) separately. .. The digestion products were electro-phoresed and purified from agarose gel by Bioneer DNA extraction kit (South Korea).

    Methylation:

    Article Title: Loss of prostasin (PRSS8) in human bladder transitional cell carcinoma cell lines is associated with epithelial-mesenchymal transition (EMT)
    Article Snippet: Paragraph title: Analysis of prostasin promoter CpG methylation state by methylation-specific PCR (MSP) ... For each cell line, aliquots of 2 μg of DNA were separately digested with 5 units of Xho I ( X ), Hpa II ( H ), or Msp I ( M ) (Invitrogen) in a total volume of 20 μl at 37°C for overnight.

    Isolation:

    Article Title: A Simple and Universal System for Gene Manipulation in Aspergillus fumigatus: In Vitro-Assembled Cas9-Guide RNA Ribonucleoproteins Coupled with Microhomology Repair Templates
    Article Snippet: The genomic DNA of 6 arbitrarily selected white colonies, which were transformed with 2 µg of the 35-bp-flanked HygR repair template, was isolated using a standard phenol-chloroform extraction protocol. .. Following quanti fication by a NanoDrop spectrophotometer and integrity verification by agarose gel electrophoresis, the genomic DNA was digested overnight at 37°C using the restriction enzyme XhoI and was separated on an agarose gel before being transferred to a Biodyne B modified nylon membrane (Thermo Scientific).

    Article Title: Plant-derived SAC domain of PAR-4 (Prostate Apoptosis Response 4) exhibits growth inhibitory effects in prostate cancer cells
    Article Snippet: .. Briefly, 10 μg of gDNA isolated from four transgenic (L2, L3, L5, and L6 of SAC-Par-4-GFP) and vector control lines were digested overnight with Xho I (Fermentas) at 37°C, subsequently electrophoresed on 0.8% TAE-agarose and transferred to a nylon membrane (Hybond-N+, Amersham). .. Blotted DNA fragments were hybridized with PCR-amplified P32 -labeled SAC-Par-4 probe using primers listed in Table .

    Size-exclusion Chromatography:

    Article Title: Bacterial Contig Map of the 21q11 Region Associated with Alzheimer's Disease and Abnormal Myelopoiesis in Down Syndrome
    Article Snippet: PAC DNA (0.50–1 μg) was digested at 37°C with one or a combination of the following restriction enzymes, Not I, Sal I (NEB), Xho I (Life Technologies). .. Electrophoresis conditions were as follows: For the 4- to 200-kb window, 5.2 V/cm, 14 hr, switch time 3–15 sec; for the 100- to 1600-kb window, 6 V/cm, 22 hr, switch time 40–80 sec. DNA size markers used (see Fig. , from left to right) were Lambda ladder (Bio-Rad), high molecular weight DNA markers (Life Technologies), 1-kb DNA ladder (Life Technologies).

    Labeling:

    Article Title: A secretory hexokinase plays an active role in the proliferation of Nosema bombycis
    Article Snippet: The above vectors and pIZ/V5-His (Thermo Fisher, Santa Clara, CA, USA) were digested by Kpn I and Xho I (Thermo Fisher, CA, USA). .. Three days later, the transfected cells nuclei were labeled with Hoechest (Thermo Fisher, Santa Clara, CA, USA).

    Article Title: A Simple and Universal System for Gene Manipulation in Aspergillus fumigatus: In Vitro-Assembled Cas9-Guide RNA Ribonucleoproteins Coupled with Microhomology Repair Templates
    Article Snippet: The resulting 504-bp PCR fragment was biotinylated with the North2South biotin random primer labeling kit (Thermo Scientific), according to the manufacturer’s protocols. .. Following quanti fication by a NanoDrop spectrophotometer and integrity verification by agarose gel electrophoresis, the genomic DNA was digested overnight at 37°C using the restriction enzyme XhoI and was separated on an agarose gel before being transferred to a Biodyne B modified nylon membrane (Thermo Scientific).

    Article Title: Plant-derived SAC domain of PAR-4 (Prostate Apoptosis Response 4) exhibits growth inhibitory effects in prostate cancer cells
    Article Snippet: Briefly, 10 μg of gDNA isolated from four transgenic (L2, L3, L5, and L6 of SAC-Par-4-GFP) and vector control lines were digested overnight with Xho I (Fermentas) at 37°C, subsequently electrophoresed on 0.8% TAE-agarose and transferred to a nylon membrane (Hybond-N+, Amersham). .. Blotted DNA fragments were hybridized with PCR-amplified P32 -labeled SAC-Par-4 probe using primers listed in Table .

    Article Title: Expression of Pennisetum glaucum Eukaryotic Translational Initiation Factor 4A (PgeIF4A) Confers Improved Drought, Salinity, and Oxidative Stress Tolerance in Groundnut
    Article Snippet: Copy number detection by southern blotting Genomic DNA (20 μg) from control and transformed plants (T0 ) was digested using 30 U of Xho I (Fermentas life sciences), single cutter of T-DNA and non-cutter of PgeIF4A gene. .. Biotin labeled PgeIF4A gene was used as probe.

    Purification:

    Article Title: Pluripotent State Induction in Mouse Embryonic Fibroblast Using mRNAs of Reprogramming Factors
    Article Snippet: In Vitro Transcription of mRNA To synthesize the mRNA of the inserted transcription factors (TFs), the designed recombinant plasmids were linearized by Xho I, followed by in vitro RNA transcription (IVT) using the mMESSAGE mMACHINE T7 kit (AM1345, Ambion® , Grand Island, NY, USA) that allows 5' cap and poly (A) tail formation. .. The transcribed mRNA was then purified using MEGAclear™ Kit (AM1908, Ambion® , Grand Island, NY, USA) according to the protocol of the manufacturer.

    Article Title: Periplasmic Expression of TNF Related Apoptosis Inducing Ligand (TRAIL) in E.coli
    Article Snippet: .. In the next step, the PCR product was purified and digested with Nde I and Xho I (Fermentas, Lithuania) and then ligated to the corresponding sites of the expression vector pET22b (Novagen, U.S) using T4 DNA ligase (Fermentas, Lithuania) ( ). .. The resulting plasmid was named OmpA-TRAIL/pET22b and the correct cloning was confirmed by PCR and sequencing.

    Article Title: RNA interference targeting virion core protein ORF095 inhibits Goatpox virus replication in Vero cells
    Article Snippet: PCR products were digested with Xho I and Bam HI, and cloned into the pEGFP-N1 expression vector (Invitrogen, Inc., Shanghai, China). .. Plasmids used for transfection were purified with the Wizard Purefection TM Plasmid DNA Purification System (Promega, USA) and quantified by Biophotometer (Eppendorf, Germany).

    Article Title: Human endogenous retroviral syncytin exerts inhibitory effect on invasive phenotype of B16F10 melanoma cells
    Article Snippet: The purified PCR products were ligated into pEGFP-N1 vector after being digested with Xho I and Eco R I. Ligations were transformed into Escherichia coli DH5α and 10 bacterial colonies were picked from per primer pair and incubated with shaking overnight in 5 mL LB medium containing 34 µg/mL kanamycin. .. The plasmids that contained the desired inserts were identified by double restriction enzyme digestion with Xho I and Eco R I, and DNA sequencing performed by Invitrogen (Guangzhou, China).

    Article Title: Exposition of hepatitis B surface antigen (HBsAg) on the surface of HEK293T cell and evaluation of its expression
    Article Snippet: The extracted plasmid of one of the resulted clones and pcDNA 3.1 Hygro (+) plasmid (Invitrogen, USA) were digested by NheI and XhoI restriction enzymes (Thermoscience, USA) separately. .. The digestion products were electro-phoresed and purified from agarose gel by Bioneer DNA extraction kit (South Korea).

    Polymerase Chain Reaction:

    Article Title: Construction of Eukaryotic Expression Vector with mBD1-mBD3 Fusion Genes and Exploring Its Activity against Influenza A Virus
    Article Snippet: .. The inserted sequences were confirmed by PCR, restriction enzyme digestion analysis with Eco R I and Xho I and sequencing using the ABI 377 DNA Analyzer (Invitrogen, Shanghai, China). .. Screening of Stable Expression MDCK Stains Transfected with pcDNA3.1(+)/mBD1-mBD3 The final concentration of MDCK cells were adjusted to 2 × 105 –5 × 105 /mL with DMEM (Gibco, Grand Island, NY, USA) supplemented with 10% bovine serum, 100 U/mL penicillin and 100 µg/mL streptomycin.

    Article Title: Loss of prostasin (PRSS8) in human bladder transitional cell carcinoma cell lines is associated with epithelial-mesenchymal transition (EMT)
    Article Snippet: Paragraph title: Analysis of prostasin promoter CpG methylation state by methylation-specific PCR (MSP) ... For each cell line, aliquots of 2 μg of DNA were separately digested with 5 units of Xho I ( X ), Hpa II ( H ), or Msp I ( M ) (Invitrogen) in a total volume of 20 μl at 37°C for overnight.

    Article Title: Cloning, Expression, and Purification of Hyperthermophile α-Amylase from Pyrococcus woesei
    Article Snippet: .. P. woesei chromosomal DNA was used as a source of the α-amylase gene and polymerase chain reaction (PCR) amplification was done with the following primers: 5′- GCTAGC TTGGAGCTTGAAGAGGGAG-3′ and 5′- GAGACC ACAATAACTCCATACGGAG-3′ containing recognition sites for restriction endonucleases Nhe I and Xho I (Fermentas; Vilnius, Lithuania). .. The reaction was performed using 10 ng DNA, 2 μL (10 μM) of each primer, 5 μL (10mM) dNTPs, 5 μL 10× PCR buffer [100mM Tris-HCl (pH 8.9), 500mM KCl, 20mM MgCl2 , 1% Triton X-100] and High-fidelity PCR Enzyme Mix (Fermentas).

    Article Title: A secretory hexokinase plays an active role in the proliferation of Nosema bombycis
    Article Snippet: The recombinant pCR-Blunt II-TOPO vector contained the targets fragments, were extracted from E. coli DH5 α . .. The above vectors and pIZ/V5-His (Thermo Fisher, Santa Clara, CA, USA) were digested by Kpn I and Xho I (Thermo Fisher, CA, USA).

    Article Title: A Simple and Universal System for Gene Manipulation in Aspergillus fumigatus: In Vitro-Assembled Cas9-Guide RNA Ribonucleoproteins Coupled with Microhomology Repair Templates
    Article Snippet: The resulting 504-bp PCR fragment was biotinylated with the North2South biotin random primer labeling kit (Thermo Scientific), according to the manufacturer’s protocols. .. Following quanti fication by a NanoDrop spectrophotometer and integrity verification by agarose gel electrophoresis, the genomic DNA was digested overnight at 37°C using the restriction enzyme XhoI and was separated on an agarose gel before being transferred to a Biodyne B modified nylon membrane (Thermo Scientific).

    Article Title: Development of a polymerase chain reaction-restriction fragment length polymorphism method for identification of the Fusarium genus using the transcription elongation factor-1α gene
    Article Snippet: Therefore, double digestion with two restriction enzymes, Xho I and Sdu I (Thermo Fisher Scientific, Inc., Waltham, MA, USA) was used for discrimination. .. The restriction digestion reaction was performed in a total volume of 20 µl containing 5 units of each enzyme, 2 µl Buffer O (Thermo Fisher Scientific, Inc.), 5 µl PCR product, and Ultrapure water (CinnaGen, Karaj, Iran) to reach a volume of 20 µl.

    Article Title: Periplasmic Expression of TNF Related Apoptosis Inducing Ligand (TRAIL) in E.coli
    Article Snippet: .. In the next step, the PCR product was purified and digested with Nde I and Xho I (Fermentas, Lithuania) and then ligated to the corresponding sites of the expression vector pET22b (Novagen, U.S) using T4 DNA ligase (Fermentas, Lithuania) ( ). .. The resulting plasmid was named OmpA-TRAIL/pET22b and the correct cloning was confirmed by PCR and sequencing.

    Article Title: RNA interference targeting virion core protein ORF095 inhibits Goatpox virus replication in Vero cells
    Article Snippet: .. PCR products were digested with Xho I and Bam HI, and cloned into the pEGFP-N1 expression vector (Invitrogen, Inc., Shanghai, China). .. The final construct p095/EGFP (Figure ) was analyzed by restriction digestion and sequenced.

    Article Title: Plant-derived SAC domain of PAR-4 (Prostate Apoptosis Response 4) exhibits growth inhibitory effects in prostate cancer cells
    Article Snippet: Briefly, 10 μg of gDNA isolated from four transgenic (L2, L3, L5, and L6 of SAC-Par-4-GFP) and vector control lines were digested overnight with Xho I (Fermentas) at 37°C, subsequently electrophoresed on 0.8% TAE-agarose and transferred to a nylon membrane (Hybond-N+, Amersham). .. Blotted DNA fragments were hybridized with PCR-amplified P32 -labeled SAC-Par-4 probe using primers listed in Table .

    Article Title: Human endogenous retroviral syncytin exerts inhibitory effect on invasive phenotype of B16F10 melanoma cells
    Article Snippet: The purified PCR products were ligated into pEGFP-N1 vector after being digested with Xho I and Eco R I. Ligations were transformed into Escherichia coli DH5α and 10 bacterial colonies were picked from per primer pair and incubated with shaking overnight in 5 mL LB medium containing 34 µg/mL kanamycin. .. The plasmids that contained the desired inserts were identified by double restriction enzyme digestion with Xho I and Eco R I, and DNA sequencing performed by Invitrogen (Guangzhou, China).

    Staining:

    Article Title: Development of a polymerase chain reaction-restriction fragment length polymorphism method for identification of the Fusarium genus using the transcription elongation factor-1α gene
    Article Snippet: Therefore, double digestion with two restriction enzymes, Xho I and Sdu I (Thermo Fisher Scientific, Inc., Waltham, MA, USA) was used for discrimination. .. Digested PCR products were electrophoresed at 50 V for 3 h on 2% agarose gel in TAE buffer and stained with ethidium bromide.

    Activated Clotting Time Assay:

    Article Title: Loss of prostasin (PRSS8) in human bladder transitional cell carcinoma cell lines is associated with epithelial-mesenchymal transition (EMT)
    Article Snippet: For each cell line, aliquots of 2 μg of DNA were separately digested with 5 units of Xho I ( X ), Hpa II ( H ), or Msp I ( M ) (Invitrogen) in a total volume of 20 μl at 37°C for overnight. .. MSP was carried out with 100 ng of the digested DNA using the following primers specific to the prostasin promoter region: upstream - 5'-CAC ATA CAC ACT ACA CA C CG -3'; and down-stream - 5'-TGG CTG CAC CTA CCT GCC CG-3'.

    Plasmid Preparation:

    Article Title: Construction of Eukaryotic Expression Vector with mBD1-mBD3 Fusion Genes and Exploring Its Activity against Influenza A Virus
    Article Snippet: Paragraph title: 2.2. The Construction of Recombinant Plasmid pcDNA3.1(+)/mBD1-mBD3 ... The inserted sequences were confirmed by PCR, restriction enzyme digestion analysis with Eco R I and Xho I and sequencing using the ABI 377 DNA Analyzer (Invitrogen, Shanghai, China).

    Article Title: Cloning, Expression, and Purification of Hyperthermophile α-Amylase from Pyrococcus woesei
    Article Snippet: Paragraph title: Construction of expression plasmid ... P. woesei chromosomal DNA was used as a source of the α-amylase gene and polymerase chain reaction (PCR) amplification was done with the following primers: 5′- GCTAGC TTGGAGCTTGAAGAGGGAG-3′ and 5′- GAGACC ACAATAACTCCATACGGAG-3′ containing recognition sites for restriction endonucleases Nhe I and Xho I (Fermentas; Vilnius, Lithuania).

    Article Title: A secretory hexokinase plays an active role in the proliferation of Nosema bombycis
    Article Snippet: The recombinant pCR-Blunt II-TOPO vector contained the targets fragments, were extracted from E. coli DH5 α . .. The above vectors and pIZ/V5-His (Thermo Fisher, Santa Clara, CA, USA) were digested by Kpn I and Xho I (Thermo Fisher, CA, USA).

    Article Title: Periplasmic Expression of TNF Related Apoptosis Inducing Ligand (TRAIL) in E.coli
    Article Snippet: .. In the next step, the PCR product was purified and digested with Nde I and Xho I (Fermentas, Lithuania) and then ligated to the corresponding sites of the expression vector pET22b (Novagen, U.S) using T4 DNA ligase (Fermentas, Lithuania) ( ). .. The resulting plasmid was named OmpA-TRAIL/pET22b and the correct cloning was confirmed by PCR and sequencing.

    Article Title: RNA interference targeting virion core protein ORF095 inhibits Goatpox virus replication in Vero cells
    Article Snippet: .. PCR products were digested with Xho I and Bam HI, and cloned into the pEGFP-N1 expression vector (Invitrogen, Inc., Shanghai, China). .. The final construct p095/EGFP (Figure ) was analyzed by restriction digestion and sequenced.

    Article Title: Plant-derived SAC domain of PAR-4 (Prostate Apoptosis Response 4) exhibits growth inhibitory effects in prostate cancer cells
    Article Snippet: .. Briefly, 10 μg of gDNA isolated from four transgenic (L2, L3, L5, and L6 of SAC-Par-4-GFP) and vector control lines were digested overnight with Xho I (Fermentas) at 37°C, subsequently electrophoresed on 0.8% TAE-agarose and transferred to a nylon membrane (Hybond-N+, Amersham). .. Blotted DNA fragments were hybridized with PCR-amplified P32 -labeled SAC-Par-4 probe using primers listed in Table .

    Article Title: Human endogenous retroviral syncytin exerts inhibitory effect on invasive phenotype of B16F10 melanoma cells
    Article Snippet: The purified PCR products were ligated into pEGFP-N1 vector after being digested with Xho I and Eco R I. Ligations were transformed into Escherichia coli DH5α and 10 bacterial colonies were picked from per primer pair and incubated with shaking overnight in 5 mL LB medium containing 34 µg/mL kanamycin. .. The plasmids that contained the desired inserts were identified by double restriction enzyme digestion with Xho I and Eco R I, and DNA sequencing performed by Invitrogen (Guangzhou, China).

    Article Title: Exposition of hepatitis B surface antigen (HBsAg) on the surface of HEK293T cell and evaluation of its expression
    Article Snippet: .. The extracted plasmid of one of the resulted clones and pcDNA 3.1 Hygro (+) plasmid (Invitrogen, USA) were digested by NheI and XhoI restriction enzymes (Thermoscience, USA) separately. .. The digestion products were electro-phoresed and purified from agarose gel by Bioneer DNA extraction kit (South Korea).

    Affinity Chromatography:

    Article Title: Periplasmic Expression of TNF Related Apoptosis Inducing Ligand (TRAIL) in E.coli
    Article Snippet: In the next step, the PCR product was purified and digested with Nde I and Xho I (Fermentas, Lithuania) and then ligated to the corresponding sites of the expression vector pET22b (Novagen, U.S) using T4 DNA ligase (Fermentas, Lithuania) ( ). .. In this expression system, the TRAIL is expressed as fused to a C-terminal poly-histidine tag, thereby making a simple one-step purification of the fusion protein by Ni-NTA affinity chromatography possible.

    Agarose Gel Electrophoresis:

    Article Title: A Simple and Universal System for Gene Manipulation in Aspergillus fumigatus: In Vitro-Assembled Cas9-Guide RNA Ribonucleoproteins Coupled with Microhomology Repair Templates
    Article Snippet: .. Following quanti fication by a NanoDrop spectrophotometer and integrity verification by agarose gel electrophoresis, the genomic DNA was digested overnight at 37°C using the restriction enzyme XhoI and was separated on an agarose gel before being transferred to a Biodyne B modified nylon membrane (Thermo Scientific). .. Membrane-bound, digested genomic DNA was hybridized with the biotinylated probe and then labeled with horseradish peroxidase-conjugated streptavidin (Thermo Scientific).

    Article Title: Development of a polymerase chain reaction-restriction fragment length polymorphism method for identification of the Fusarium genus using the transcription elongation factor-1α gene
    Article Snippet: Therefore, double digestion with two restriction enzymes, Xho I and Sdu I (Thermo Fisher Scientific, Inc., Waltham, MA, USA) was used for discrimination. .. Digested PCR products were electrophoresed at 50 V for 3 h on 2% agarose gel in TAE buffer and stained with ethidium bromide.

    Article Title: Human endogenous retroviral syncytin exerts inhibitory effect on invasive phenotype of B16F10 melanoma cells
    Article Snippet: The PCR products of 1.65 kb were excised after agarose gel electrophoresis and purified by QIAquick Gel Extraction Kit (QIAGEN, Germany). .. The plasmids that contained the desired inserts were identified by double restriction enzyme digestion with Xho I and Eco R I, and DNA sequencing performed by Invitrogen (Guangzhou, China).

    Article Title: Exposition of hepatitis B surface antigen (HBsAg) on the surface of HEK293T cell and evaluation of its expression
    Article Snippet: The extracted plasmid of one of the resulted clones and pcDNA 3.1 Hygro (+) plasmid (Invitrogen, USA) were digested by NheI and XhoI restriction enzymes (Thermoscience, USA) separately. .. The digestion products were electro-phoresed and purified from agarose gel by Bioneer DNA extraction kit (South Korea).

    Article Title: Expression of Pennisetum glaucum Eukaryotic Translational Initiation Factor 4A (PgeIF4A) Confers Improved Drought, Salinity, and Oxidative Stress Tolerance in Groundnut
    Article Snippet: Copy number detection by southern blotting Genomic DNA (20 μg) from control and transformed plants (T0 ) was digested using 30 U of Xho I (Fermentas life sciences), single cutter of T-DNA and non-cutter of PgeIF4A gene. .. The digested samples were size fractionated on agarose gel (0.8%) for 16 h at 30 V. The depurinated and denatured DNA fragments were subsequently transferred to positively charged Hybond N+ nylon membrane (GE Health Care, USA).

    In Vitro:

    Article Title: Pluripotent State Induction in Mouse Embryonic Fibroblast Using mRNAs of Reprogramming Factors
    Article Snippet: .. In Vitro Transcription of mRNA To synthesize the mRNA of the inserted transcription factors (TFs), the designed recombinant plasmids were linearized by Xho I, followed by in vitro RNA transcription (IVT) using the mMESSAGE mMACHINE T7 kit (AM1345, Ambion® , Grand Island, NY, USA) that allows 5' cap and poly (A) tail formation. .. The transcribed mRNA was then purified using MEGAclear™ Kit (AM1908, Ambion® , Grand Island, NY, USA) according to the protocol of the manufacturer.

    Article Title: Development and Application of a Reverse Genetics System for Japanese Encephalitis Virus
    Article Snippet: RNA transcripts were synthesized by in vitro transcription. .. Here, 100 to 200 ng of the template DNA linearized with Xho I or Xba I digestion and in some cases modified with mung bean nuclease was added to a 25-μl reaction mixture consisting of the buffer supplied by the manufacturer (Gibco-BRL) plus 0.6 mM cap analog [m7 G(5′)ppp(5′)A or m7 G(5′)ppp(5′)G; NEB Inc.], 0.5 μM [3 H]UTP (1.0 mCi/ml, 50 Ci/mmol; New England Nuclear Corp., Boston, Mass.), 10 mM DTT, 1 mM each UTP, GTP, CTP, and ATP, 40 U of RNaseOUT, and 15 U of SP6 RNA polymerase (Gibco-BRL).

    Transgenic Assay:

    Article Title: Plant-derived SAC domain of PAR-4 (Prostate Apoptosis Response 4) exhibits growth inhibitory effects in prostate cancer cells
    Article Snippet: .. Briefly, 10 μg of gDNA isolated from four transgenic (L2, L3, L5, and L6 of SAC-Par-4-GFP) and vector control lines were digested overnight with Xho I (Fermentas) at 37°C, subsequently electrophoresed on 0.8% TAE-agarose and transferred to a nylon membrane (Hybond-N+, Amersham). .. Blotted DNA fragments were hybridized with PCR-amplified P32 -labeled SAC-Par-4 probe using primers listed in Table .

    Spectrophotometry:

    Article Title: A Simple and Universal System for Gene Manipulation in Aspergillus fumigatus: In Vitro-Assembled Cas9-Guide RNA Ribonucleoproteins Coupled with Microhomology Repair Templates
    Article Snippet: .. Following quanti fication by a NanoDrop spectrophotometer and integrity verification by agarose gel electrophoresis, the genomic DNA was digested overnight at 37°C using the restriction enzyme XhoI and was separated on an agarose gel before being transferred to a Biodyne B modified nylon membrane (Thermo Scientific). .. Membrane-bound, digested genomic DNA was hybridized with the biotinylated probe and then labeled with horseradish peroxidase-conjugated streptavidin (Thermo Scientific).

    Article Title: Pluripotent State Induction in Mouse Embryonic Fibroblast Using mRNAs of Reprogramming Factors
    Article Snippet: In Vitro Transcription of mRNA To synthesize the mRNA of the inserted transcription factors (TFs), the designed recombinant plasmids were linearized by Xho I, followed by in vitro RNA transcription (IVT) using the mMESSAGE mMACHINE T7 kit (AM1345, Ambion® , Grand Island, NY, USA) that allows 5' cap and poly (A) tail formation. .. The mRNA concentration was measured using a NanoDrop 1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA).

    Concentration Assay:

    Article Title: Pluripotent State Induction in Mouse Embryonic Fibroblast Using mRNAs of Reprogramming Factors
    Article Snippet: In Vitro Transcription of mRNA To synthesize the mRNA of the inserted transcription factors (TFs), the designed recombinant plasmids were linearized by Xho I, followed by in vitro RNA transcription (IVT) using the mMESSAGE mMACHINE T7 kit (AM1345, Ambion® , Grand Island, NY, USA) that allows 5' cap and poly (A) tail formation. .. The mRNA concentration was measured using a NanoDrop 1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA).

    DNA Purification:

    Article Title: RNA interference targeting virion core protein ORF095 inhibits Goatpox virus replication in Vero cells
    Article Snippet: PCR products were digested with Xho I and Bam HI, and cloned into the pEGFP-N1 expression vector (Invitrogen, Inc., Shanghai, China). .. Plasmids used for transfection were purified with the Wizard Purefection TM Plasmid DNA Purification System (Promega, USA) and quantified by Biophotometer (Eppendorf, Germany).

    CTG Assay:

    Article Title: Loss of prostasin (PRSS8) in human bladder transitional cell carcinoma cell lines is associated with epithelial-mesenchymal transition (EMT)
    Article Snippet: For each cell line, aliquots of 2 μg of DNA were separately digested with 5 units of Xho I ( X ), Hpa II ( H ), or Msp I ( M ) (Invitrogen) in a total volume of 20 μl at 37°C for overnight. .. MSP was carried out with 100 ng of the digested DNA using the following primers specific to the prostasin promoter region: upstream - 5'-CAC ATA CAC ACT ACA CA C CG -3'; and down-stream - 5'-TGG CTG CAC CTA CCT GCC CG-3'.

    Article Title: Human endogenous retroviral syncytin exerts inhibitory effect on invasive phenotype of B16F10 melanoma cells
    Article Snippet: The forward primer 5'-CGCG CTCGAG GCCAccATGGCCCTCCCTTATCATATTTTTCTCTTTA CTG-3' (containing an Xho I site) and reverse primer 5'-AATC GAATT CGACTGCTTCCTGCTGAATTGG-3' (containing an Eco R I site) were used to amplify human syncytin cDNA. .. The plasmids that contained the desired inserts were identified by double restriction enzyme digestion with Xho I and Eco R I, and DNA sequencing performed by Invitrogen (Guangzhou, China).

    Gel Extraction:

    Article Title: Human endogenous retroviral syncytin exerts inhibitory effect on invasive phenotype of B16F10 melanoma cells
    Article Snippet: The PCR products of 1.65 kb were excised after agarose gel electrophoresis and purified by QIAquick Gel Extraction Kit (QIAGEN, Germany). .. The plasmids that contained the desired inserts were identified by double restriction enzyme digestion with Xho I and Eco R I, and DNA sequencing performed by Invitrogen (Guangzhou, China).

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    Thermo Fisher xhoi restriction enzymes
    (A) Digestion on extracted plasmid of one of positive clones with NheI and <t>XhoI</t> confirmed HBsAg fragment insertion in <t>pcDNA</t> vector. Column 1: Mix DNA ladder (Thermoscientific, USA). Column 2: Undigested circular plasmid. Column 3: 696 bp and 5501 bp bands that confirmed cloning. (B) Digestion of recombinant plasmid with BglII enzyme. Column 1: Mix DNA ladder (Thermoscientific, USA). Column 2: Undigested plasmid. Column 3: A 6197 bp linearized plasmid.
    Xhoi Restriction Enzymes, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    (A) Digestion on extracted plasmid of one of positive clones with NheI and XhoI confirmed HBsAg fragment insertion in pcDNA vector. Column 1: Mix DNA ladder (Thermoscientific, USA). Column 2: Undigested circular plasmid. Column 3: 696 bp and 5501 bp bands that confirmed cloning. (B) Digestion of recombinant plasmid with BglII enzyme. Column 1: Mix DNA ladder (Thermoscientific, USA). Column 2: Undigested plasmid. Column 3: A 6197 bp linearized plasmid.

    Journal: Research in Pharmaceutical Sciences

    Article Title: Exposition of hepatitis B surface antigen (HBsAg) on the surface of HEK293T cell and evaluation of its expression

    doi: 10.4103/1735-5362.192485

    Figure Lengend Snippet: (A) Digestion on extracted plasmid of one of positive clones with NheI and XhoI confirmed HBsAg fragment insertion in pcDNA vector. Column 1: Mix DNA ladder (Thermoscientific, USA). Column 2: Undigested circular plasmid. Column 3: 696 bp and 5501 bp bands that confirmed cloning. (B) Digestion of recombinant plasmid with BglII enzyme. Column 1: Mix DNA ladder (Thermoscientific, USA). Column 2: Undigested plasmid. Column 3: A 6197 bp linearized plasmid.

    Article Snippet: The extracted plasmid of one of the resulted clones and pcDNA 3.1 Hygro (+) plasmid (Invitrogen, USA) were digested by NheI and XhoI restriction enzymes (Thermoscience, USA) separately.

    Techniques: Plasmid Preparation, Clone Assay, Recombinant

    Southern blot analysis of Δ pksP mutant generated in the Af293 background. (A) Schematic representation of the genomic locus of the Af293 and Δ pksP strains. Deletion of the pksP gene was carried out using the HygR cassette. The cleavage sites of the dual in vitro -assembled Cas9 RNPs are marked by thick vertical lines. XhoI cutting sites are indicated in the pksP locus of the wild-type and Δ pksP strains. (B) Southern blot analysis of 6 arbitrarily selected colonies after digesting genomic DNA with the XhoI restriction enzyme. The wild type (WT) produced a 1.8-kb band that matches the expected wild-type banding pattern. Lanes 1, 2, 4, 5, and 6 displayed a 3.8-kb band which matches the expected pksP deletion banding pattern. The colony in lane 3 displayed a 7.6-kb band, likely containing a tandem integration of the HygR repair template at the pksP locus.

    Journal: mSphere

    Article Title: A Simple and Universal System for Gene Manipulation in Aspergillus fumigatus: In Vitro-Assembled Cas9-Guide RNA Ribonucleoproteins Coupled with Microhomology Repair Templates

    doi: 10.1128/mSphere.00446-17

    Figure Lengend Snippet: Southern blot analysis of Δ pksP mutant generated in the Af293 background. (A) Schematic representation of the genomic locus of the Af293 and Δ pksP strains. Deletion of the pksP gene was carried out using the HygR cassette. The cleavage sites of the dual in vitro -assembled Cas9 RNPs are marked by thick vertical lines. XhoI cutting sites are indicated in the pksP locus of the wild-type and Δ pksP strains. (B) Southern blot analysis of 6 arbitrarily selected colonies after digesting genomic DNA with the XhoI restriction enzyme. The wild type (WT) produced a 1.8-kb band that matches the expected wild-type banding pattern. Lanes 1, 2, 4, 5, and 6 displayed a 3.8-kb band which matches the expected pksP deletion banding pattern. The colony in lane 3 displayed a 7.6-kb band, likely containing a tandem integration of the HygR repair template at the pksP locus.

    Article Snippet: Following quanti fication by a NanoDrop spectrophotometer and integrity verification by agarose gel electrophoresis, the genomic DNA was digested overnight at 37°C using the restriction enzyme XhoI and was separated on an agarose gel before being transferred to a Biodyne B modified nylon membrane (Thermo Scientific).

    Techniques: Southern Blot, Mutagenesis, Generated, In Vitro, Produced