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    New England Biolabs ecor1 new england biolabs
    Ecor1 New England Biolabs, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs ecori new england biolabs
    Representative variation in MSAP profiles. “→” to red arrows represents variation in DNA methylation between diploid and tetraploid plants; “+” represents fragments obtained after digestion with <t>EcoR</t> I or Hpa II/ Msp I; “−” represents fragments not digested by EcoR I or Hpa II/ Msp I; Type I fragments are nonmethylated and were presented in both the H ( EcoR I or Hpa II digest) and M ( EcoR I or Msp I digest) lanes; Type II are fully methylated and only appeared in the M lanes; Type III are hemimethylated and appeared in the H lanes; Type IV were fragments absent from both H and M lanes in diploid but present in either H or M lane of tetraploid, and vice versa.
    Ecori New England Biolabs, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1186 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs 3195l t4 dna ligase reaction buffer new england biolabs cat
    Representative variation in MSAP profiles. “→” to red arrows represents variation in DNA methylation between diploid and tetraploid plants; “+” represents fragments obtained after digestion with <t>EcoR</t> I or Hpa II/ Msp I; “−” represents fragments not digested by EcoR I or Hpa II/ Msp I; Type I fragments are nonmethylated and were presented in both the H ( EcoR I or Hpa II digest) and M ( EcoR I or Msp I digest) lanes; Type II are fully methylated and only appeared in the M lanes; Type III are hemimethylated and appeared in the H lanes; Type IV were fragments absent from both H and M lanes in diploid but present in either H or M lane of tetraploid, and vice versa.
    3195l T4 Dna Ligase Reaction Buffer New England Biolabs Cat, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs ecor
    Site-specific integration at the rDNA locus of MSCs. (a) Schematic of the construction of pHr2-NL. pHr2-NL contained two inverted expression cassettes, one consisting of an IRES element from the encephalomyocarditis virus, the coding region of the Neo gene, the SV40 polyA signal (SV40pA), and two loxP sites with the same orientation. LoxP sites were recognized by CRE enzyme to remove the Neo cassette after gene targeting. LHA, long homologous arm (U13369:937-6523); SHA, short homologous arm (U13369:6523–7643). The genomic locus indicates the 6.7 kb fragment (U13369:937-7643) required for homologous recombination at the internal transcribed spacer 1 (ITS1) of the rRNA gene. Single cutting sites for restricted enzymes of Nco I, <t>EcoR</t> I, Hind III , and <t>Pvu</t> II are located at the IRES-Neo frame and outside of the long homologous fragment. The fragment between the two Pvu II sites was 8285 bp in size, and it was detected using probe 1 (P1). The expected sizes of the restriction fragments produced by Nco I, EcoR I, and Hind III were 4001 bp, 7628 bp, and 15,316 bp, respectively. These were detected using probe 2 (P2). Primer t-up was located at the SV40 polyA. Primer t-re was located outside of the SHA at the hrDNA locus. (b) Drug-resistant cell in basal medium. (c) Drug-resistant colonies in the medium supplemented with VEGF+bFGF+Vc+ITS-X. (d) Identification of colonies with site-specific integration by PCR. The expected fragment, 1.3 kb in size, was amplified from the genomic DNA of colonies using site-specific integration. M, DL200 DNA marker; 1, negative colony; 2–5, positive colonies; 6, wild-type MSCs. (e–f) Southern blotting analysis of the representative recombinants. Genomic DNA digested with Pvu II , Nco I, EcoR I, and Hind III was analyzed. A specific band was consistently detected in colonies 1-1, 1-2, 2-1, and 2-2. An additional band beside the specific band was detected in colony 2-3. c, control (untransfected MSCs); N, Nco I; E, EcoR I; H, Hind III .
    Ecor, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 107 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    TaKaRa ecorv
    Site-specific integration at the rDNA locus of MSCs. (a) Schematic of the construction of pHr2-NL. pHr2-NL contained two inverted expression cassettes, one consisting of an IRES element from the encephalomyocarditis virus, the coding region of the Neo gene, the SV40 polyA signal (SV40pA), and two loxP sites with the same orientation. LoxP sites were recognized by CRE enzyme to remove the Neo cassette after gene targeting. LHA, long homologous arm (U13369:937-6523); SHA, short homologous arm (U13369:6523–7643). The genomic locus indicates the 6.7 kb fragment (U13369:937-7643) required for homologous recombination at the internal transcribed spacer 1 (ITS1) of the rRNA gene. Single cutting sites for restricted enzymes of Nco I, <t>EcoR</t> I, Hind III , and <t>Pvu</t> II are located at the IRES-Neo frame and outside of the long homologous fragment. The fragment between the two Pvu II sites was 8285 bp in size, and it was detected using probe 1 (P1). The expected sizes of the restriction fragments produced by Nco I, EcoR I, and Hind III were 4001 bp, 7628 bp, and 15,316 bp, respectively. These were detected using probe 2 (P2). Primer t-up was located at the SV40 polyA. Primer t-re was located outside of the SHA at the hrDNA locus. (b) Drug-resistant cell in basal medium. (c) Drug-resistant colonies in the medium supplemented with VEGF+bFGF+Vc+ITS-X. (d) Identification of colonies with site-specific integration by PCR. The expected fragment, 1.3 kb in size, was amplified from the genomic DNA of colonies using site-specific integration. M, DL200 DNA marker; 1, negative colony; 2–5, positive colonies; 6, wild-type MSCs. (e–f) Southern blotting analysis of the representative recombinants. Genomic DNA digested with Pvu II , Nco I, EcoR I, and Hind III was analyzed. A specific band was consistently detected in colonies 1-1, 1-2, 2-1, and 2-2. An additional band beside the specific band was detected in colony 2-3. c, control (untransfected MSCs); N, Nco I; E, EcoR I; H, Hind III .
    Ecorv, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 477 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs ecor i hf
    Proof-of-concept assembly of 16TU construct. (A) A schematic showing the four intermediate Level 2 constructs for the assembly of the 16-TU construct. The carotenoid biosynthesis genes crtE , crtB , crtI , and crtY assembled in the Vector A, the yellow chromoprotein genes scOrange , amilGFP , amajLime , and fwYellow in the Vector B, the pink chromoprotein genes tsPurple , eforRed , spisPink , and mRFP1 in the Vector Γ, and the violacein biosynthesis genes vioA , vioB , vioD and vioE in the Vector Δ. (B) A schematic of the 16TU construct derived from the assembly of the four Level 2 cassettes, each containing 4-TUs, in the Level 1 Acceptor Vector A. (C) Cells transformed with the successfully assembled 16TU construct grew into black colonies due to predominant colouring by protoviolaceinic acid. (D) Gel electrophoresis of six plasmids (isolated from the black colonies) digested with <t>PstI</t> and <t>EcoRI</t> resulting in bands of expected sizes—18.2kb for the insert and 2.2kb for the vector. (E) The same plasmids were digested with PstI and AleI resulting in the bands of expected sizes—7.1kb, 5.1 and 4.9kb (appear merged on the gel), and 3.2kb.
    Ecor I Hf, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 84 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs bamhi ecori
    Replication fork leading strands progress up to the psoralen crosslink. (A) Sequence of the plasmid around the psoralen ICL with restriction sites used in B and C. Double arrows indicate the size of the replicated strands (lagging or leading) spanning the ICL site for the control or repaired plasmid. Single arrows indicate the progression of the leading strand to the ICL for the crosslinked plasmid. The strand size is shown above each product. (B) Mapping of leading strand progression for pTUC and pTUC-PSO after 65 min of incubation in Xenopus egg extracts in the continuous presence of [α- 32 P]-dATP. Plasmids were digested by the indicated enzymes and analyzed on 10% polyacrylamide denaturing gel. Faint bands visible in lanes 3 and 4 likely result from a star activity of Sac II. The two 28 nt size indicators are at two different positions due to gel smiling. (C) Mapping of leading strand progression for pTUC and pTUC-PSO at 25, 35, 50, 65, 85, 95, 120 and 180 min. Plasmids were digested with <t>EcoRI</t> and <t>BamHI</t> and subjected to migration on a 10% polyacrylamide denaturing gel. 28 nt and 46 nt fragments correspond to the DNA size expected for the leading strand arriving at the psoralen from the EcoRI side or BamHI side, respectively.
    Bamhi Ecori, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 88 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Representative variation in MSAP profiles. “→” to red arrows represents variation in DNA methylation between diploid and tetraploid plants; “+” represents fragments obtained after digestion with EcoR I or Hpa II/ Msp I; “−” represents fragments not digested by EcoR I or Hpa II/ Msp I; Type I fragments are nonmethylated and were presented in both the H ( EcoR I or Hpa II digest) and M ( EcoR I or Msp I digest) lanes; Type II are fully methylated and only appeared in the M lanes; Type III are hemimethylated and appeared in the H lanes; Type IV were fragments absent from both H and M lanes in diploid but present in either H or M lane of tetraploid, and vice versa.

    Journal: International Journal of Molecular Sciences

    Article Title: Morphological, Genome and Gene Expression Changes in Newly Induced Autopolyploid Chrysanthemum lavandulifolium (Fisch. ex Trautv.) Makino

    doi: 10.3390/ijms17101690

    Figure Lengend Snippet: Representative variation in MSAP profiles. “→” to red arrows represents variation in DNA methylation between diploid and tetraploid plants; “+” represents fragments obtained after digestion with EcoR I or Hpa II/ Msp I; “−” represents fragments not digested by EcoR I or Hpa II/ Msp I; Type I fragments are nonmethylated and were presented in both the H ( EcoR I or Hpa II digest) and M ( EcoR I or Msp I digest) lanes; Type II are fully methylated and only appeared in the M lanes; Type III are hemimethylated and appeared in the H lanes; Type IV were fragments absent from both H and M lanes in diploid but present in either H or M lane of tetraploid, and vice versa.

    Article Snippet: Mthylation Sensitive Amplified Polymorphism Analysis The MSAP technique was applied to the DNA pools from three diploid lines and three tetraploid lines C. lavandulifolium They were digested with either EcoR I and Hpa II or EcoR I and Msp I (NEB) at 37 °C for 12 h. The digested fragments were ligated to 5 pmol EcoR I adaptor and 50 pmol Hpa II/Msp I adaptor by incubation with 4 U T4 DNA polymerase (NEB) at 16 °C for 4 has described for the AFLP method.

    Techniques: DNA Methylation Assay, Methylation

    Repair of MB + VL-induced 8-oxoG from the Ad-encoded lacZ gene in human and rodent cells measured by loss of Fpg-sensitive sites. ( A ) Southern blot analysis of the repair of MB + VL-induced 8-oxoG in the Ad lacZ gene. Shown here is a representative blot. Lanes 1 and 2 contain untreated Ad DNA, while lanes 3 and 4 contain Ad DNA exposed to 480 s VL in phosphate buffer with 20 mg/ml MB. Lanes 1–4 have not undergone any repair incubation. The presence of ssDNA breaks in the 3-kb EcoRI lacZ fragment produce smaller ssDNA fragments that migrate further than the full-length fragment. These smaller fragments appear as a smear or tail below the defined 3-kb band. Smearing below the 3-kb band in samples that have not been treated with Fpg (lanes 1 and 3) represent ssDNA breaks from other sources. It can be seen that a small amount of Fpg-sensitive 8-oxoG lesions are present prior to treatment with MB + VL (compare lanes 1 and 2). Following MB + VL exposure, a large number of Fpg-sensitive sites are generated (compare lanes 2 and 4). During repair incubation, BER removes 8-oxoG resulting in the loss of T4pdg-sensitive sites and recovery of the full-length 3-kb lacZ fragment. As long as 8-oxoG lesions persist in the lacZ DNA, Fpg will induce ssDNA breaks resulting in fewer full-length fragments and less signal compared to the control. ( B ) Quantification of the percent removal of Fpg-sensitive sites from the Ad-encoded lacZ gene in GM637F and CHO-AA8 cells. Each point on the graphs represents the arithmetic mean ± SE of the percent removal of MB + VL-induced Fpg-sensitive sites from three independent experiments. A significant increase in the percent removal of MB + VL-induced Fpg-sensitive sites was observed in GM637F at 24 h (indicated by an asterisk) and a significant difference in the percent removal of Fpg-sensitive sites was observed between GM637F and CHO-AA8 at 24 h (indicated by a cross/plus sign).

    Journal: Mutagenesis

    Article Title: Host cell reactivation of gene expression for an adenovirus-encoded reporter gene reflects the repair of UVC-induced cyclobutane pyrimidine dimers and methylene blue plus visible light-induced 8-oxoguanine

    doi: 10.1093/mutage/get027

    Figure Lengend Snippet: Repair of MB + VL-induced 8-oxoG from the Ad-encoded lacZ gene in human and rodent cells measured by loss of Fpg-sensitive sites. ( A ) Southern blot analysis of the repair of MB + VL-induced 8-oxoG in the Ad lacZ gene. Shown here is a representative blot. Lanes 1 and 2 contain untreated Ad DNA, while lanes 3 and 4 contain Ad DNA exposed to 480 s VL in phosphate buffer with 20 mg/ml MB. Lanes 1–4 have not undergone any repair incubation. The presence of ssDNA breaks in the 3-kb EcoRI lacZ fragment produce smaller ssDNA fragments that migrate further than the full-length fragment. These smaller fragments appear as a smear or tail below the defined 3-kb band. Smearing below the 3-kb band in samples that have not been treated with Fpg (lanes 1 and 3) represent ssDNA breaks from other sources. It can be seen that a small amount of Fpg-sensitive 8-oxoG lesions are present prior to treatment with MB + VL (compare lanes 1 and 2). Following MB + VL exposure, a large number of Fpg-sensitive sites are generated (compare lanes 2 and 4). During repair incubation, BER removes 8-oxoG resulting in the loss of T4pdg-sensitive sites and recovery of the full-length 3-kb lacZ fragment. As long as 8-oxoG lesions persist in the lacZ DNA, Fpg will induce ssDNA breaks resulting in fewer full-length fragments and less signal compared to the control. ( B ) Quantification of the percent removal of Fpg-sensitive sites from the Ad-encoded lacZ gene in GM637F and CHO-AA8 cells. Each point on the graphs represents the arithmetic mean ± SE of the percent removal of MB + VL-induced Fpg-sensitive sites from three independent experiments. A significant increase in the percent removal of MB + VL-induced Fpg-sensitive sites was observed in GM637F at 24 h (indicated by an asterisk) and a significant difference in the percent removal of Fpg-sensitive sites was observed between GM637F and CHO-AA8 at 24 h (indicated by a cross/plus sign).

    Article Snippet: Prior to treatment of Ad DNA with either T4 pyrimidine-DNA glycosylase [T4pdg; New England Biolabs (NEB) M0308S] or formamidopyrimidine (Fapy)-DNA glycosylase (Fpg; NEB M02040), all samples were digested overnight by 40 units of EcoRI (NEB R0101) in a total reaction volume of 50 µl in 1× NEB buffer 1.

    Techniques: Southern Blot, Incubation, Generated

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

    Journal: Journal of Clinical Microbiology

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

    doi: 10.1128/JCM.02340-10

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

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

    Techniques: Transfection

    The Δ recA strain is transformable when it is complemented. a recA complementation plasmid pJGW92. The hatched region was derived from C. bescii native plasmid pBAS2. apr R apramycin resistance casette, cat R thiamphenicol resistance casette, repA replication initiation protein for the E. coli pSC101 replication origin, par partitioning locus for E. coli . Restriction sites for structural verification are shown on the plasmid map. b Restriction digests with AvaI and EcoRI. The expected bands from AvaI are 5.1, 2.6, and 1.1 kb. The expected bands from EcoRI are 6.9 and 1.9 kb. + purified pJGW92 from E. coli . Lanes 1–3: plasmids isolated from E. coli transformed with DNA isolated from JWCT26 (Δ recA + pJGW92). c ∆ recA strains transformed with complementation plasmids retain the chromosomal deletion of recA . Primers indicated in the gene diagram were used to amplify DNA extracted from C. thermocellum strains. d JG161 and DC232 verified the replace-ment of recA by the C. bescii pyrF gene. Two different primer pairs (JG161/JG155 and JG162/ JG144) were used to ensure that the transformed strain retained the recA deletion. PCR was performed for both 30 cycles (30X) and 40 cycles (40X) using primer pair JG162/JG144. The molecular weight ladder in kilobases for all gels is shown

    Journal: Journal of industrial microbiology & biotechnology

    Article Title: Deletion of the Clostridium thermocellum recA gene reveals that it is required for thermophilic plasmid replication but not plasmid integration at homologous DNA sequences

    doi: 10.1007/s10295-018-2049-x

    Figure Lengend Snippet: The Δ recA strain is transformable when it is complemented. a recA complementation plasmid pJGW92. The hatched region was derived from C. bescii native plasmid pBAS2. apr R apramycin resistance casette, cat R thiamphenicol resistance casette, repA replication initiation protein for the E. coli pSC101 replication origin, par partitioning locus for E. coli . Restriction sites for structural verification are shown on the plasmid map. b Restriction digests with AvaI and EcoRI. The expected bands from AvaI are 5.1, 2.6, and 1.1 kb. The expected bands from EcoRI are 6.9 and 1.9 kb. + purified pJGW92 from E. coli . Lanes 1–3: plasmids isolated from E. coli transformed with DNA isolated from JWCT26 (Δ recA + pJGW92). c ∆ recA strains transformed with complementation plasmids retain the chromosomal deletion of recA . Primers indicated in the gene diagram were used to amplify DNA extracted from C. thermocellum strains. d JG161 and DC232 verified the replace-ment of recA by the C. bescii pyrF gene. Two different primer pairs (JG161/JG155 and JG162/ JG144) were used to ensure that the transformed strain retained the recA deletion. PCR was performed for both 30 cycles (30X) and 40 cycles (40X) using primer pair JG162/JG144. The molecular weight ladder in kilobases for all gels is shown

    Article Snippet: The colonies were picked into 10 mL LB with 50 μg/mL apramycin, and the plasmid DNA was extracted using a Miniprep kit (Qiagen, Valencia, CA, USA) and screened with restriction enzymes EcoRI and AvaI for pJGW92 and NcoI and AvaI for pJGW93 (NEB).

    Techniques: Plasmid Preparation, Derivative Assay, Purification, Isolation, Transformation Assay, Polymerase Chain Reaction, Molecular Weight

    Site-specific integration at the rDNA locus of MSCs. (a) Schematic of the construction of pHr2-NL. pHr2-NL contained two inverted expression cassettes, one consisting of an IRES element from the encephalomyocarditis virus, the coding region of the Neo gene, the SV40 polyA signal (SV40pA), and two loxP sites with the same orientation. LoxP sites were recognized by CRE enzyme to remove the Neo cassette after gene targeting. LHA, long homologous arm (U13369:937-6523); SHA, short homologous arm (U13369:6523–7643). The genomic locus indicates the 6.7 kb fragment (U13369:937-7643) required for homologous recombination at the internal transcribed spacer 1 (ITS1) of the rRNA gene. Single cutting sites for restricted enzymes of Nco I, EcoR I, Hind III , and Pvu II are located at the IRES-Neo frame and outside of the long homologous fragment. The fragment between the two Pvu II sites was 8285 bp in size, and it was detected using probe 1 (P1). The expected sizes of the restriction fragments produced by Nco I, EcoR I, and Hind III were 4001 bp, 7628 bp, and 15,316 bp, respectively. These were detected using probe 2 (P2). Primer t-up was located at the SV40 polyA. Primer t-re was located outside of the SHA at the hrDNA locus. (b) Drug-resistant cell in basal medium. (c) Drug-resistant colonies in the medium supplemented with VEGF+bFGF+Vc+ITS-X. (d) Identification of colonies with site-specific integration by PCR. The expected fragment, 1.3 kb in size, was amplified from the genomic DNA of colonies using site-specific integration. M, DL200 DNA marker; 1, negative colony; 2–5, positive colonies; 6, wild-type MSCs. (e–f) Southern blotting analysis of the representative recombinants. Genomic DNA digested with Pvu II , Nco I, EcoR I, and Hind III was analyzed. A specific band was consistently detected in colonies 1-1, 1-2, 2-1, and 2-2. An additional band beside the specific band was detected in colony 2-3. c, control (untransfected MSCs); N, Nco I; E, EcoR I; H, Hind III .

    Journal: BioMed Research International

    Article Title: Nonviral Gene Targeting at rDNA Locus of Human Mesenchymal Stem Cells

    doi: 10.1155/2013/135189

    Figure Lengend Snippet: Site-specific integration at the rDNA locus of MSCs. (a) Schematic of the construction of pHr2-NL. pHr2-NL contained two inverted expression cassettes, one consisting of an IRES element from the encephalomyocarditis virus, the coding region of the Neo gene, the SV40 polyA signal (SV40pA), and two loxP sites with the same orientation. LoxP sites were recognized by CRE enzyme to remove the Neo cassette after gene targeting. LHA, long homologous arm (U13369:937-6523); SHA, short homologous arm (U13369:6523–7643). The genomic locus indicates the 6.7 kb fragment (U13369:937-7643) required for homologous recombination at the internal transcribed spacer 1 (ITS1) of the rRNA gene. Single cutting sites for restricted enzymes of Nco I, EcoR I, Hind III , and Pvu II are located at the IRES-Neo frame and outside of the long homologous fragment. The fragment between the two Pvu II sites was 8285 bp in size, and it was detected using probe 1 (P1). The expected sizes of the restriction fragments produced by Nco I, EcoR I, and Hind III were 4001 bp, 7628 bp, and 15,316 bp, respectively. These were detected using probe 2 (P2). Primer t-up was located at the SV40 polyA. Primer t-re was located outside of the SHA at the hrDNA locus. (b) Drug-resistant cell in basal medium. (c) Drug-resistant colonies in the medium supplemented with VEGF+bFGF+Vc+ITS-X. (d) Identification of colonies with site-specific integration by PCR. The expected fragment, 1.3 kb in size, was amplified from the genomic DNA of colonies using site-specific integration. M, DL200 DNA marker; 1, negative colony; 2–5, positive colonies; 6, wild-type MSCs. (e–f) Southern blotting analysis of the representative recombinants. Genomic DNA digested with Pvu II , Nco I, EcoR I, and Hind III was analyzed. A specific band was consistently detected in colonies 1-1, 1-2, 2-1, and 2-2. An additional band beside the specific band was detected in colony 2-3. c, control (untransfected MSCs); N, Nco I; E, EcoR I; H, Hind III .

    Article Snippet: Southern Blotting After overnight digestion with restriction enzymes Pvu II, Nco I, EcoR I, and Hind III (New England Biolabs, Ipswich, MA, USA), 3 μ g of genomic DNA per sample was electrophoresed on a 0.8% agarose gel overnight and then transferred to positively charged nylon membranes (Hybond-N+, Amersham, Piscataway, NJ, USA).

    Techniques: Expressing, Homologous Recombination, Produced, Polymerase Chain Reaction, Amplification, Marker, Southern Blot

    Confirmation of the mutation in PA14Δ hepP . PA14 and PA14Δ hepP were grown in LB broth and the chromosomal DNA was extracted. a PCR analysis to detect the presence of MAR2xT7 within hepP . PCR reactions were run using the chromosomal DNA from each strain as a template and primers corresponding to the DNA sequences 94 bp upstream and 179 bp downstream of the hepP structural gene ( zbdP- For3/ hepP- Rev3, Table 2 ). The expected 1926-bp fragment from PA14 (lane 1) and the 2920-bp fragment (the additional 994 bp from MAR2xT7 ) from PA14Δ hepP (lane 2) were detected. Lane 3 is a no-template control and the molecular size standards are in lane 4. b Restriction analysis of the PCR products. The coding sequence for hepP does not contain an EcoR V restriction enzyme site, while MAR2xT7 contains a single EcoR V site. Digestion of the PCR products with EcoR V failed to reduce the size of the 1926-bp fragment obtained from PA14 (lane 3) but resulted in the cleavage of the product obtained from PA14Δ hepP into the expected 800 bp and 2120 bp fragments (lane 4). Lane 1 contains the molecular size standards; lane 2 was left empty

    Journal: BMC Microbiology

    Article Title: Isolation and characterization of HepP: a virulence-related Pseudomonas aeruginosa heparinase

    doi: 10.1186/s12866-017-1141-0

    Figure Lengend Snippet: Confirmation of the mutation in PA14Δ hepP . PA14 and PA14Δ hepP were grown in LB broth and the chromosomal DNA was extracted. a PCR analysis to detect the presence of MAR2xT7 within hepP . PCR reactions were run using the chromosomal DNA from each strain as a template and primers corresponding to the DNA sequences 94 bp upstream and 179 bp downstream of the hepP structural gene ( zbdP- For3/ hepP- Rev3, Table 2 ). The expected 1926-bp fragment from PA14 (lane 1) and the 2920-bp fragment (the additional 994 bp from MAR2xT7 ) from PA14Δ hepP (lane 2) were detected. Lane 3 is a no-template control and the molecular size standards are in lane 4. b Restriction analysis of the PCR products. The coding sequence for hepP does not contain an EcoR V restriction enzyme site, while MAR2xT7 contains a single EcoR V site. Digestion of the PCR products with EcoR V failed to reduce the size of the 1926-bp fragment obtained from PA14 (lane 3) but resulted in the cleavage of the product obtained from PA14Δ hepP into the expected 800 bp and 2120 bp fragments (lane 4). Lane 1 contains the molecular size standards; lane 2 was left empty

    Article Snippet: Therefore, to further confirm the mutation of hepP in PA14ΔhepP , we performed restriction enzyme digestion on the PCR products with EcoR V (New England Biolabs).

    Techniques: Mutagenesis, Polymerase Chain Reaction, Sequencing

    Controlled release of USPIO micelles by environmental triggers . (A) Self-assembly of EcoRV-sensitive ssDNA-USPIO clusters, and subsequent enzymatic treatment results in measurable changes in R 2 relaxation coefficient relative to initial values. Following EcoRV treatment, R 2 values return to baseline, a phenomenon that is in significant contrast to the effects of EcoRI treatment of the same clusters (n = 6). * p

    Journal: Journal of Nanobiotechnology

    Article Title: Enzymatic- and temperature-sensitive controlled release of ultrasmall superparamagnetic iron oxides (USPIOs)

    doi: 10.1186/1477-3155-9-7

    Figure Lengend Snippet: Controlled release of USPIO micelles by environmental triggers . (A) Self-assembly of EcoRV-sensitive ssDNA-USPIO clusters, and subsequent enzymatic treatment results in measurable changes in R 2 relaxation coefficient relative to initial values. Following EcoRV treatment, R 2 values return to baseline, a phenomenon that is in significant contrast to the effects of EcoRI treatment of the same clusters (n = 6). * p

    Article Snippet: The restriction enzymes EcoRI and EcoRV were purchased from New England Biolabs (Ipswich, MA).

    Techniques:

    Proof-of-concept assembly of 16TU construct. (A) A schematic showing the four intermediate Level 2 constructs for the assembly of the 16-TU construct. The carotenoid biosynthesis genes crtE , crtB , crtI , and crtY assembled in the Vector A, the yellow chromoprotein genes scOrange , amilGFP , amajLime , and fwYellow in the Vector B, the pink chromoprotein genes tsPurple , eforRed , spisPink , and mRFP1 in the Vector Γ, and the violacein biosynthesis genes vioA , vioB , vioD and vioE in the Vector Δ. (B) A schematic of the 16TU construct derived from the assembly of the four Level 2 cassettes, each containing 4-TUs, in the Level 1 Acceptor Vector A. (C) Cells transformed with the successfully assembled 16TU construct grew into black colonies due to predominant colouring by protoviolaceinic acid. (D) Gel electrophoresis of six plasmids (isolated from the black colonies) digested with PstI and EcoRI resulting in bands of expected sizes—18.2kb for the insert and 2.2kb for the vector. (E) The same plasmids were digested with PstI and AleI resulting in the bands of expected sizes—7.1kb, 5.1 and 4.9kb (appear merged on the gel), and 3.2kb.

    Journal: PLoS ONE

    Article Title: Mobius Assembly: A versatile Golden-Gate framework towards universal DNA assembly

    doi: 10.1371/journal.pone.0189892

    Figure Lengend Snippet: Proof-of-concept assembly of 16TU construct. (A) A schematic showing the four intermediate Level 2 constructs for the assembly of the 16-TU construct. The carotenoid biosynthesis genes crtE , crtB , crtI , and crtY assembled in the Vector A, the yellow chromoprotein genes scOrange , amilGFP , amajLime , and fwYellow in the Vector B, the pink chromoprotein genes tsPurple , eforRed , spisPink , and mRFP1 in the Vector Γ, and the violacein biosynthesis genes vioA , vioB , vioD and vioE in the Vector Δ. (B) A schematic of the 16TU construct derived from the assembly of the four Level 2 cassettes, each containing 4-TUs, in the Level 1 Acceptor Vector A. (C) Cells transformed with the successfully assembled 16TU construct grew into black colonies due to predominant colouring by protoviolaceinic acid. (D) Gel electrophoresis of six plasmids (isolated from the black colonies) digested with PstI and EcoRI resulting in bands of expected sizes—18.2kb for the insert and 2.2kb for the vector. (E) The same plasmids were digested with PstI and AleI resulting in the bands of expected sizes—7.1kb, 5.1 and 4.9kb (appear merged on the gel), and 3.2kb.

    Article Snippet: Subsequently the pSB1C3/pSB1K3 backbones and the Golden Gate cassettes were digested with EcoRI-HF and PstI-HF (NEB) for 20 min at 37°C followed by purification with PCR Clean-up kit (Macherey Nagel).

    Techniques: Construct, Plasmid Preparation, Derivative Assay, Transformation Assay, Nucleic Acid Electrophoresis, Isolation

    Replication fork leading strands progress up to the psoralen crosslink. (A) Sequence of the plasmid around the psoralen ICL with restriction sites used in B and C. Double arrows indicate the size of the replicated strands (lagging or leading) spanning the ICL site for the control or repaired plasmid. Single arrows indicate the progression of the leading strand to the ICL for the crosslinked plasmid. The strand size is shown above each product. (B) Mapping of leading strand progression for pTUC and pTUC-PSO after 65 min of incubation in Xenopus egg extracts in the continuous presence of [α- 32 P]-dATP. Plasmids were digested by the indicated enzymes and analyzed on 10% polyacrylamide denaturing gel. Faint bands visible in lanes 3 and 4 likely result from a star activity of Sac II. The two 28 nt size indicators are at two different positions due to gel smiling. (C) Mapping of leading strand progression for pTUC and pTUC-PSO at 25, 35, 50, 65, 85, 95, 120 and 180 min. Plasmids were digested with EcoRI and BamHI and subjected to migration on a 10% polyacrylamide denaturing gel. 28 nt and 46 nt fragments correspond to the DNA size expected for the leading strand arriving at the psoralen from the EcoRI side or BamHI side, respectively.

    Journal: PLoS ONE

    Article Title: Replication-Fork Stalling and Processing at a Single Psoralen Interstrand Crosslink in Xenopus Egg Extracts

    doi: 10.1371/journal.pone.0018554

    Figure Lengend Snippet: Replication fork leading strands progress up to the psoralen crosslink. (A) Sequence of the plasmid around the psoralen ICL with restriction sites used in B and C. Double arrows indicate the size of the replicated strands (lagging or leading) spanning the ICL site for the control or repaired plasmid. Single arrows indicate the progression of the leading strand to the ICL for the crosslinked plasmid. The strand size is shown above each product. (B) Mapping of leading strand progression for pTUC and pTUC-PSO after 65 min of incubation in Xenopus egg extracts in the continuous presence of [α- 32 P]-dATP. Plasmids were digested by the indicated enzymes and analyzed on 10% polyacrylamide denaturing gel. Faint bands visible in lanes 3 and 4 likely result from a star activity of Sac II. The two 28 nt size indicators are at two different positions due to gel smiling. (C) Mapping of leading strand progression for pTUC and pTUC-PSO at 25, 35, 50, 65, 85, 95, 120 and 180 min. Plasmids were digested with EcoRI and BamHI and subjected to migration on a 10% polyacrylamide denaturing gel. 28 nt and 46 nt fragments correspond to the DNA size expected for the leading strand arriving at the psoralen from the EcoRI side or BamHI side, respectively.

    Article Snippet: Denaturing gel electrophoretic analysis of crosslinked products Crosslinked samples were digested with BamHI + EcoRI (New England Biolabs), filled with [α-32 P]-dATP using AMV reverse transcriptase as described by the manufacturer (Finnzyme).

    Techniques: Sequencing, Plasmid Preparation, Incubation, Activity Assay, Migration

    TFO and purification of monomodified plasmid. (A) Structure of the TFO linked in 3′ to 4, 5′, 8-trimethylpsoralen and in 5′ to biotin. (B) Localization of the TFO binding site and position of the psoralen ICL on pTUC plasmid. P and A are schematic representations of primers for q-PCR used in (D). P primers surround the psoralen ICL site and amplify a region of 113 nt, the A primers amplify an undamaged regions of 129 nt. (C) Analysis of the crosslinked plasmid after UV irradiation and before (input) or after (purified) DTT elution from a strepatvidin column. DNA was digested with BamHI + EcoRI and radioactively labelled before electrophoresis on a 10% polyacrylamide denaturing gel. Interpretative diagrams of the relevant molecular species are shown on lane sides. (D) Input and purified plasmids were subjected to q-PCR with primers P and A. Mean values of 3 q-PCRs for the input and 12 q-PCRs for the purified plasmid are presented. For calculation details see Material and Methods . (E) After purification pTUC-PSO was analyzed on a 0.8% agarose gel TBE 1x alongside with control plasmid and a molecular weight ladder.

    Journal: PLoS ONE

    Article Title: Replication-Fork Stalling and Processing at a Single Psoralen Interstrand Crosslink in Xenopus Egg Extracts

    doi: 10.1371/journal.pone.0018554

    Figure Lengend Snippet: TFO and purification of monomodified plasmid. (A) Structure of the TFO linked in 3′ to 4, 5′, 8-trimethylpsoralen and in 5′ to biotin. (B) Localization of the TFO binding site and position of the psoralen ICL on pTUC plasmid. P and A are schematic representations of primers for q-PCR used in (D). P primers surround the psoralen ICL site and amplify a region of 113 nt, the A primers amplify an undamaged regions of 129 nt. (C) Analysis of the crosslinked plasmid after UV irradiation and before (input) or after (purified) DTT elution from a strepatvidin column. DNA was digested with BamHI + EcoRI and radioactively labelled before electrophoresis on a 10% polyacrylamide denaturing gel. Interpretative diagrams of the relevant molecular species are shown on lane sides. (D) Input and purified plasmids were subjected to q-PCR with primers P and A. Mean values of 3 q-PCRs for the input and 12 q-PCRs for the purified plasmid are presented. For calculation details see Material and Methods . (E) After purification pTUC-PSO was analyzed on a 0.8% agarose gel TBE 1x alongside with control plasmid and a molecular weight ladder.

    Article Snippet: Denaturing gel electrophoretic analysis of crosslinked products Crosslinked samples were digested with BamHI + EcoRI (New England Biolabs), filled with [α-32 P]-dATP using AMV reverse transcriptase as described by the manufacturer (Finnzyme).

    Techniques: Purification, Plasmid Preparation, Binding Assay, Polymerase Chain Reaction, Irradiation, Electrophoresis, Agarose Gel Electrophoresis, Molecular Weight