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  • 98
    New England Biolabs clai
    Transduction of hES cells with murine retroviral vectors. ( A ) Transfection efficiencies of undifferentiated hES cells using optimized protocols for lipofection (L), electroporation (E) and nucleofection (N) techniques. ( B ) mCAT1-HA expression (green) in nucleofected hES cells cultured on matrigel. Twenty-four hours after plating, the cells show a flattened morphology typical for colonies propagated on matrigel. Nuclear expression of Oct-4 (red) reflects their undifferentiated state. ( C ) Schematic illustration of the two-step protocol used for transduction of hES cells with ecotropic retroviral vectors. First cells are nucleofected with a construct encoding the murine retrovirus receptor mCAT1. Twenty-four hours later they are transduced with a murine retroviral vector. Transduced cultures are either analyzed for transgene expression after 48 h or subjected to selection of permanently transduced clones. The integrated provirus expresses the EGFP transgene from the viral LTR linked to a neomycin resistance gene (neoR) by an internal ribosome entry site (IRES). ( D ) Forty-eight hours after infection, 16.4 ± 5.9% of the total cell population showed EGFP-expression. Normalized to the proportion of mCAT1-expressing cells determined in (A), this corresponds to a calculated transduction efficiency of ∼30% of the mCAT1-expressing cells (mean values from n = 5 independent experiments). ( E – G ) Transduced EGFP-positive cells continue to express the pluripotency-associated markers Oct-4 (E), Tra-1-60 (F) and Tra-1-81 (G) (all red; counterstain DAPI). ( H ) Five passages after transduction, four clones were subjected to Southern analysis. A single integration of the EGFP transgene could be detected in clones PK2, PK4 and PK7. Clone PK5 displays two bands, which could be the result of a double integration, a mixed clone population or a mutation of the vector provirus. Restriction analysis was performed with <t>EcoRI</t> (for genomic DNA) and <t>ClaI</t> (unique site within the retroviral vector). Scale bars: B,E: 100 μm; F: 30 μm; G: 50 μm.
    Clai, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 98/100, based on 507 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Millipore clai
    Two-dimensional gel electrophoresis analysis of rDNA replication intermediates following removal of HU. Cells were synchronized at the G1/S border by starvation and cultured in growth media in the absence or presence of 20 mM HU. <t>DNA</t> was prepared from mock-treated starved cells, mock-treated S phase cells, and HU-treated cells at defined intervals following HU removal. (A) Upper diagram: schematic of the 21 kb rDNA minichromosome and location of relevant restriction sites and probes for Southern blot analysis. Macronuclear rDNA minichromosomes contain two copies of the rRNA coding region and adjacent 5' and 3 'non-transcribed spacer (NTS) regions in an inverted orientation. The 35S rRNA precursor encodes the 17S, 5.8S and 26S rRNAs (grey areas- mature RNA coding regions, black and white stippled areas- processed RNA precursor regions, hatched area- self-splicing 26S rRNA intron). Telomeric DNA repeats (vertical hashes) are present at chromosome termini. The positions of four probes for N/N and N/A 2D gel analysis are shown. Expanded view of the 1.9 kb 5 NTS. Thick arrow- rRNA promoter; grey ovals- position of phase nucleosomes in vegetative rDNA minichromosomes [ 60 ], black boxes- type 1 repeats. Domains 1 and 2 (thin arrows) are 430 bp imperfect tandem repeats with 230 bp nuclease hypersensitive regions Lower diagram: schematic of typical RI patterns detected by N/N 2D gel electrophoresis. Simple Y arc (arrow): passive replication of the probed DNA fragment interval due to initiation elsewhere in the chromosome. Bubble arc (arrowhead): initiation at a central position in the probed DNA fragment. Bubble-to-Y arc: initiation at an asymmetric position in the examined fragment (low MW RIs: bubble arc (arrowhead), high MW RIs: Y arc). Composite: active (complete bubble arc, arrowhead) and passive (complete Y arc, arrow) replication within the probed DNA fragment. X and Double Y: the X spike (arrowhead) is generated from branch migration recombinant intermediates, and the double Y (open arrow) is generated by two converging replication forks. (B) RI patterns detected with the 5’ NTS probe 1 on HindIII digested DNA from mock treated quiescent (starvation) and replicating cell populations (S phase). (C) 5’ NTS analysis on DNA from HU-treated cells 0–120 min after HU removal (arrow: simple Y arc, passive replication; arrowhead: X-spike recombination intermediates). See flow cytometry profiles ( Fig 5A ) and western blots ( Fig 5B ) for cell cycle progression and abundance of Orc1p and Mcm6p, respectively. (D) Two-dimensional N-N gel analysis of the 5.5 kb rRNA coding region <t>ClaI</t> fragment (position 2168–7629, probe 2). (E) Two dimensional neutral-alkaline (N/A) analysis of RIs derived from the rRNA coding region ClaI fragment. Probe 3 spans nucleotides 2169 to 3670, and probe 4 spans nucleotides 5214–6676. Schematic of nascent-strand RIs resolved by N/A 2D gel electrophoresis. The 1n spot corresponds to non-replicating DNA. The vertical smear is derived from nicked, non-replicating DNA, and the horizontal smear represents the parental strand in RIs of different length. The diagonal arc corresponds to nascent-strand replication intermediates that are liberated from the parental strand by alkali denaturation prior to electrophoresis in the second dimension.
    Clai, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 66 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Promega clai
    Organization of the PDE1 Locus and Targeted Gene Disruption. (A) Restriction map of the insertional mutant locus of pde1 ) are indicated ( HPH ). pCB1003 integration was found to have occurred 165 bp upstream of a large open reading frame (gray shading). <t>ClaI</t> sites found on either side of the integrated plasmid were used to excise a fragment of the insertion locus. (B) Restriction map of the PDE1 locus isolated as a 6.94-kb PstI-SalI fragment from a Magnaporthe strain Guy11 genomic library. The PDE1 locus contains a 4575-bp open reading frame interrupted by a single intron at positions 3927 to 3997. The arrow indicates the orientation of the open reading frame and the position of the intron. (C) Targeted gene disruption vector pPde1::Hph. The vector was made by excising a 2.6-kb PstI fragment at the 5′ end of PDE1 and inserting the hygromycin B resistance gene cassette ( HPH ) in a SmaI site. (D) Restriction map of the pde1 :: Hph disruption allele showing the position of the hygromycin resistance gene cassette ( HPH ) at the 5′ end of the PDE1 open reading frame. (E) <t>DNA</t> gel blot analysis of pPde1::Hph transformants. Genomic DNA was prepared from the wild-type strain Guy11 (lane 1), the ectopic integration transformant PV9 (lane 2), and four pde1 :: Hph mutant transformants, PV1, PV2, PV3, and PV4 (lanes 3 to 6, respectively). Genomic DNA was digested with PstI and separated on a 0.8% agarose gel. The blot was probed with pSKPS1. The Xs between (B) and (C) indicate a crossover event. B, BamHI; C, ClaI; P, PstI; Sa, SalI; Sm, SmaI; X, XhoI.
    Clai, supplied by Promega, used in various techniques. Bioz Stars score: 92/100, based on 128 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Thermo Fisher fastdigest clai
    Organization of the PDE1 Locus and Targeted Gene Disruption. (A) Restriction map of the insertional mutant locus of pde1 ) are indicated ( HPH ). pCB1003 integration was found to have occurred 165 bp upstream of a large open reading frame (gray shading). <t>ClaI</t> sites found on either side of the integrated plasmid were used to excise a fragment of the insertion locus. (B) Restriction map of the PDE1 locus isolated as a 6.94-kb PstI-SalI fragment from a Magnaporthe strain Guy11 genomic library. The PDE1 locus contains a 4575-bp open reading frame interrupted by a single intron at positions 3927 to 3997. The arrow indicates the orientation of the open reading frame and the position of the intron. (C) Targeted gene disruption vector pPde1::Hph. The vector was made by excising a 2.6-kb PstI fragment at the 5′ end of PDE1 and inserting the hygromycin B resistance gene cassette ( HPH ) in a SmaI site. (D) Restriction map of the pde1 :: Hph disruption allele showing the position of the hygromycin resistance gene cassette ( HPH ) at the 5′ end of the PDE1 open reading frame. (E) <t>DNA</t> gel blot analysis of pPde1::Hph transformants. Genomic DNA was prepared from the wild-type strain Guy11 (lane 1), the ectopic integration transformant PV9 (lane 2), and four pde1 :: Hph mutant transformants, PV1, PV2, PV3, and PV4 (lanes 3 to 6, respectively). Genomic DNA was digested with PstI and separated on a 0.8% agarose gel. The blot was probed with pSKPS1. The Xs between (B) and (C) indicate a crossover event. B, BamHI; C, ClaI; P, PstI; Sa, SalI; Sm, SmaI; X, XhoI.
    Fastdigest Clai, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Millipore clay pipe
    Organization of the PDE1 Locus and Targeted Gene Disruption. (A) Restriction map of the insertional mutant locus of pde1 ) are indicated ( HPH ). pCB1003 integration was found to have occurred 165 bp upstream of a large open reading frame (gray shading). <t>ClaI</t> sites found on either side of the integrated plasmid were used to excise a fragment of the insertion locus. (B) Restriction map of the PDE1 locus isolated as a 6.94-kb PstI-SalI fragment from a Magnaporthe strain Guy11 genomic library. The PDE1 locus contains a 4575-bp open reading frame interrupted by a single intron at positions 3927 to 3997. The arrow indicates the orientation of the open reading frame and the position of the intron. (C) Targeted gene disruption vector pPde1::Hph. The vector was made by excising a 2.6-kb PstI fragment at the 5′ end of PDE1 and inserting the hygromycin B resistance gene cassette ( HPH ) in a SmaI site. (D) Restriction map of the pde1 :: Hph disruption allele showing the position of the hygromycin resistance gene cassette ( HPH ) at the 5′ end of the PDE1 open reading frame. (E) <t>DNA</t> gel blot analysis of pPde1::Hph transformants. Genomic DNA was prepared from the wild-type strain Guy11 (lane 1), the ectopic integration transformant PV9 (lane 2), and four pde1 :: Hph mutant transformants, PV1, PV2, PV3, and PV4 (lanes 3 to 6, respectively). Genomic DNA was digested with PstI and separated on a 0.8% agarose gel. The blot was probed with pSKPS1. The Xs between (B) and (C) indicate a crossover event. B, BamHI; C, ClaI; P, PstI; Sa, SalI; Sm, SmaI; X, XhoI.
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    91
    Millipore ecori clai
    Organization of the PDE1 Locus and Targeted Gene Disruption. (A) Restriction map of the insertional mutant locus of pde1 ) are indicated ( HPH ). pCB1003 integration was found to have occurred 165 bp upstream of a large open reading frame (gray shading). <t>ClaI</t> sites found on either side of the integrated plasmid were used to excise a fragment of the insertion locus. (B) Restriction map of the PDE1 locus isolated as a 6.94-kb PstI-SalI fragment from a Magnaporthe strain Guy11 genomic library. The PDE1 locus contains a 4575-bp open reading frame interrupted by a single intron at positions 3927 to 3997. The arrow indicates the orientation of the open reading frame and the position of the intron. (C) Targeted gene disruption vector pPde1::Hph. The vector was made by excising a 2.6-kb PstI fragment at the 5′ end of PDE1 and inserting the hygromycin B resistance gene cassette ( HPH ) in a SmaI site. (D) Restriction map of the pde1 :: Hph disruption allele showing the position of the hygromycin resistance gene cassette ( HPH ) at the 5′ end of the PDE1 open reading frame. (E) <t>DNA</t> gel blot analysis of pPde1::Hph transformants. Genomic DNA was prepared from the wild-type strain Guy11 (lane 1), the ectopic integration transformant PV9 (lane 2), and four pde1 :: Hph mutant transformants, PV1, PV2, PV3, and PV4 (lanes 3 to 6, respectively). Genomic DNA was digested with PstI and separated on a 0.8% agarose gel. The blot was probed with pSKPS1. The Xs between (B) and (C) indicate a crossover event. B, BamHI; C, ClaI; P, PstI; Sa, SalI; Sm, SmaI; X, XhoI.
    Ecori Clai, supplied by Millipore, used in various techniques. Bioz Stars score: 91/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher clai
    Organization of the PDE1 Locus and Targeted Gene Disruption. (A) Restriction map of the insertional mutant locus of pde1 ) are indicated ( HPH ). pCB1003 integration was found to have occurred 165 bp upstream of a large open reading frame (gray shading). <t>ClaI</t> sites found on either side of the integrated plasmid were used to excise a fragment of the insertion locus. (B) Restriction map of the PDE1 locus isolated as a 6.94-kb PstI-SalI fragment from a Magnaporthe strain Guy11 genomic library. The PDE1 locus contains a 4575-bp open reading frame interrupted by a single intron at positions 3927 to 3997. The arrow indicates the orientation of the open reading frame and the position of the intron. (C) Targeted gene disruption vector pPde1::Hph. The vector was made by excising a 2.6-kb PstI fragment at the 5′ end of PDE1 and inserting the hygromycin B resistance gene cassette ( HPH ) in a SmaI site. (D) Restriction map of the pde1 :: Hph disruption allele showing the position of the hygromycin resistance gene cassette ( HPH ) at the 5′ end of the PDE1 open reading frame. (E) <t>DNA</t> gel blot analysis of pPde1::Hph transformants. Genomic DNA was prepared from the wild-type strain Guy11 (lane 1), the ectopic integration transformant PV9 (lane 2), and four pde1 :: Hph mutant transformants, PV1, PV2, PV3, and PV4 (lanes 3 to 6, respectively). Genomic DNA was digested with PstI and separated on a 0.8% agarose gel. The blot was probed with pSKPS1. The Xs between (B) and (C) indicate a crossover event. B, BamHI; C, ClaI; P, PstI; Sa, SalI; Sm, SmaI; X, XhoI.
    Clai, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 370 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Transduction of hES cells with murine retroviral vectors. ( A ) Transfection efficiencies of undifferentiated hES cells using optimized protocols for lipofection (L), electroporation (E) and nucleofection (N) techniques. ( B ) mCAT1-HA expression (green) in nucleofected hES cells cultured on matrigel. Twenty-four hours after plating, the cells show a flattened morphology typical for colonies propagated on matrigel. Nuclear expression of Oct-4 (red) reflects their undifferentiated state. ( C ) Schematic illustration of the two-step protocol used for transduction of hES cells with ecotropic retroviral vectors. First cells are nucleofected with a construct encoding the murine retrovirus receptor mCAT1. Twenty-four hours later they are transduced with a murine retroviral vector. Transduced cultures are either analyzed for transgene expression after 48 h or subjected to selection of permanently transduced clones. The integrated provirus expresses the EGFP transgene from the viral LTR linked to a neomycin resistance gene (neoR) by an internal ribosome entry site (IRES). ( D ) Forty-eight hours after infection, 16.4 ± 5.9% of the total cell population showed EGFP-expression. Normalized to the proportion of mCAT1-expressing cells determined in (A), this corresponds to a calculated transduction efficiency of ∼30% of the mCAT1-expressing cells (mean values from n = 5 independent experiments). ( E – G ) Transduced EGFP-positive cells continue to express the pluripotency-associated markers Oct-4 (E), Tra-1-60 (F) and Tra-1-81 (G) (all red; counterstain DAPI). ( H ) Five passages after transduction, four clones were subjected to Southern analysis. A single integration of the EGFP transgene could be detected in clones PK2, PK4 and PK7. Clone PK5 displays two bands, which could be the result of a double integration, a mixed clone population or a mutation of the vector provirus. Restriction analysis was performed with EcoRI (for genomic DNA) and ClaI (unique site within the retroviral vector). Scale bars: B,E: 100 μm; F: 30 μm; G: 50 μm.

    Journal: Nucleic Acids Research

    Article Title: Transduction of human embryonic stem cells by ecotropic retroviral vectors

    doi: 10.1093/nar/gkl674

    Figure Lengend Snippet: Transduction of hES cells with murine retroviral vectors. ( A ) Transfection efficiencies of undifferentiated hES cells using optimized protocols for lipofection (L), electroporation (E) and nucleofection (N) techniques. ( B ) mCAT1-HA expression (green) in nucleofected hES cells cultured on matrigel. Twenty-four hours after plating, the cells show a flattened morphology typical for colonies propagated on matrigel. Nuclear expression of Oct-4 (red) reflects their undifferentiated state. ( C ) Schematic illustration of the two-step protocol used for transduction of hES cells with ecotropic retroviral vectors. First cells are nucleofected with a construct encoding the murine retrovirus receptor mCAT1. Twenty-four hours later they are transduced with a murine retroviral vector. Transduced cultures are either analyzed for transgene expression after 48 h or subjected to selection of permanently transduced clones. The integrated provirus expresses the EGFP transgene from the viral LTR linked to a neomycin resistance gene (neoR) by an internal ribosome entry site (IRES). ( D ) Forty-eight hours after infection, 16.4 ± 5.9% of the total cell population showed EGFP-expression. Normalized to the proportion of mCAT1-expressing cells determined in (A), this corresponds to a calculated transduction efficiency of ∼30% of the mCAT1-expressing cells (mean values from n = 5 independent experiments). ( E – G ) Transduced EGFP-positive cells continue to express the pluripotency-associated markers Oct-4 (E), Tra-1-60 (F) and Tra-1-81 (G) (all red; counterstain DAPI). ( H ) Five passages after transduction, four clones were subjected to Southern analysis. A single integration of the EGFP transgene could be detected in clones PK2, PK4 and PK7. Clone PK5 displays two bands, which could be the result of a double integration, a mixed clone population or a mutation of the vector provirus. Restriction analysis was performed with EcoRI (for genomic DNA) and ClaI (unique site within the retroviral vector). Scale bars: B,E: 100 μm; F: 30 μm; G: 50 μm.

    Article Snippet: DNA was digested overnight using EcoRI and ClaI (both NEB) restriction enzymes and separated on an agarose gel.

    Techniques: Transduction, Transfection, Electroporation, Expressing, Cell Culture, Construct, Plasmid Preparation, Selection, Clone Assay, Infection, Mutagenesis

    Confirmation of xer1 disruption in selected M . agalactiae ‘switchover’ clones via PCR and Southern analysis. (A) Verification of xer1 disruption in crude DNA extracts of selected ‘switchover’ clones by PCR using primers RecEndET28 and T3ISLrev specific to the chromosomal xer1 region and the pR3 plasmid backbone, respectively. 2 kb PCR product confirms xer1 disruption [ 13 ] in selected ‘switchover’ clones picked from the right (R)—parotideal (PAR) or—mandibular (MAN) lymph nodes of sheep MS 7 or MS 10. The observed bands were comparable to the positive controls corresponding to the crude DNA extracts (PLMY and PLMU) as well to pure DNA preparation of PLMY (+DNA); as expected this band was absent in the three negative controls corresponding to water (H 2 O), PG2 crude extract (PG2) and SP4 broth (Medium). (B) Southerns were performed as described earlier [ 13 ] whereby Cla I-digested genomic DNA was hybridized with xer1 -specific probe. xer1 disruption is confirmed in the ‘switchover’ clones (lanes 3–8) by the presence of two bands of 3.7 kb and ~18.9 kb as also seen for PLMU control (lane 2), whereas the wild type PG2 (lane 1) with intact xer1 shows a 13 kb band. Disruption plasmid pR3 (lane 9) shows the expected 10 kb band. Switchover clones: lane 3, MS 10 right udder/VpmaV; lane 4, MS 6 mesenterial LN/VpmaW; lane 5, MS 6 right iliac LN/VpmaX; lane 6, MS 6 mesenterial LN/VpmaW (PLM W); lane 7, MS 9 right udder/VpmaX (PLM X) ; lane 8, MS 6 right iliac LN/VpmaX.

    Journal: PLoS Pathogens

    Article Title: Vpma phase variation is important for survival and persistence of Mycoplasma agalactiae in the immunocompetent host

    doi: 10.1371/journal.ppat.1006656

    Figure Lengend Snippet: Confirmation of xer1 disruption in selected M . agalactiae ‘switchover’ clones via PCR and Southern analysis. (A) Verification of xer1 disruption in crude DNA extracts of selected ‘switchover’ clones by PCR using primers RecEndET28 and T3ISLrev specific to the chromosomal xer1 region and the pR3 plasmid backbone, respectively. 2 kb PCR product confirms xer1 disruption [ 13 ] in selected ‘switchover’ clones picked from the right (R)—parotideal (PAR) or—mandibular (MAN) lymph nodes of sheep MS 7 or MS 10. The observed bands were comparable to the positive controls corresponding to the crude DNA extracts (PLMY and PLMU) as well to pure DNA preparation of PLMY (+DNA); as expected this band was absent in the three negative controls corresponding to water (H 2 O), PG2 crude extract (PG2) and SP4 broth (Medium). (B) Southerns were performed as described earlier [ 13 ] whereby Cla I-digested genomic DNA was hybridized with xer1 -specific probe. xer1 disruption is confirmed in the ‘switchover’ clones (lanes 3–8) by the presence of two bands of 3.7 kb and ~18.9 kb as also seen for PLMU control (lane 2), whereas the wild type PG2 (lane 1) with intact xer1 shows a 13 kb band. Disruption plasmid pR3 (lane 9) shows the expected 10 kb band. Switchover clones: lane 3, MS 10 right udder/VpmaV; lane 4, MS 6 mesenterial LN/VpmaW; lane 5, MS 6 right iliac LN/VpmaX; lane 6, MS 6 mesenterial LN/VpmaW (PLM W); lane 7, MS 9 right udder/VpmaX (PLM X) ; lane 8, MS 6 right iliac LN/VpmaX.

    Article Snippet: Confirming xer1 disruption via Southern blot analyses Mycoplasma genomic DNA was isolated by QIAamp DNA Mini Kit (Qiagen) and digested with Cla I (New England Biolabs) at 37°C for at least 6–7 h before subjecting to electrophoresis on a 1% agarose gel.

    Techniques: Clone Assay, Polymerase Chain Reaction, Plasmid Preparation, Mass Spectrometry

    Two-dimensional gel electrophoresis analysis of rDNA replication intermediates following removal of HU. Cells were synchronized at the G1/S border by starvation and cultured in growth media in the absence or presence of 20 mM HU. DNA was prepared from mock-treated starved cells, mock-treated S phase cells, and HU-treated cells at defined intervals following HU removal. (A) Upper diagram: schematic of the 21 kb rDNA minichromosome and location of relevant restriction sites and probes for Southern blot analysis. Macronuclear rDNA minichromosomes contain two copies of the rRNA coding region and adjacent 5' and 3 'non-transcribed spacer (NTS) regions in an inverted orientation. The 35S rRNA precursor encodes the 17S, 5.8S and 26S rRNAs (grey areas- mature RNA coding regions, black and white stippled areas- processed RNA precursor regions, hatched area- self-splicing 26S rRNA intron). Telomeric DNA repeats (vertical hashes) are present at chromosome termini. The positions of four probes for N/N and N/A 2D gel analysis are shown. Expanded view of the 1.9 kb 5 NTS. Thick arrow- rRNA promoter; grey ovals- position of phase nucleosomes in vegetative rDNA minichromosomes [ 60 ], black boxes- type 1 repeats. Domains 1 and 2 (thin arrows) are 430 bp imperfect tandem repeats with 230 bp nuclease hypersensitive regions Lower diagram: schematic of typical RI patterns detected by N/N 2D gel electrophoresis. Simple Y arc (arrow): passive replication of the probed DNA fragment interval due to initiation elsewhere in the chromosome. Bubble arc (arrowhead): initiation at a central position in the probed DNA fragment. Bubble-to-Y arc: initiation at an asymmetric position in the examined fragment (low MW RIs: bubble arc (arrowhead), high MW RIs: Y arc). Composite: active (complete bubble arc, arrowhead) and passive (complete Y arc, arrow) replication within the probed DNA fragment. X and Double Y: the X spike (arrowhead) is generated from branch migration recombinant intermediates, and the double Y (open arrow) is generated by two converging replication forks. (B) RI patterns detected with the 5’ NTS probe 1 on HindIII digested DNA from mock treated quiescent (starvation) and replicating cell populations (S phase). (C) 5’ NTS analysis on DNA from HU-treated cells 0–120 min after HU removal (arrow: simple Y arc, passive replication; arrowhead: X-spike recombination intermediates). See flow cytometry profiles ( Fig 5A ) and western blots ( Fig 5B ) for cell cycle progression and abundance of Orc1p and Mcm6p, respectively. (D) Two-dimensional N-N gel analysis of the 5.5 kb rRNA coding region ClaI fragment (position 2168–7629, probe 2). (E) Two dimensional neutral-alkaline (N/A) analysis of RIs derived from the rRNA coding region ClaI fragment. Probe 3 spans nucleotides 2169 to 3670, and probe 4 spans nucleotides 5214–6676. Schematic of nascent-strand RIs resolved by N/A 2D gel electrophoresis. The 1n spot corresponds to non-replicating DNA. The vertical smear is derived from nicked, non-replicating DNA, and the horizontal smear represents the parental strand in RIs of different length. The diagonal arc corresponds to nascent-strand replication intermediates that are liberated from the parental strand by alkali denaturation prior to electrophoresis in the second dimension.

    Journal: PLoS Genetics

    Article Title: Checkpoint Activation of an Unconventional DNA Replication Program in Tetrahymena

    doi: 10.1371/journal.pgen.1005405

    Figure Lengend Snippet: Two-dimensional gel electrophoresis analysis of rDNA replication intermediates following removal of HU. Cells were synchronized at the G1/S border by starvation and cultured in growth media in the absence or presence of 20 mM HU. DNA was prepared from mock-treated starved cells, mock-treated S phase cells, and HU-treated cells at defined intervals following HU removal. (A) Upper diagram: schematic of the 21 kb rDNA minichromosome and location of relevant restriction sites and probes for Southern blot analysis. Macronuclear rDNA minichromosomes contain two copies of the rRNA coding region and adjacent 5' and 3 'non-transcribed spacer (NTS) regions in an inverted orientation. The 35S rRNA precursor encodes the 17S, 5.8S and 26S rRNAs (grey areas- mature RNA coding regions, black and white stippled areas- processed RNA precursor regions, hatched area- self-splicing 26S rRNA intron). Telomeric DNA repeats (vertical hashes) are present at chromosome termini. The positions of four probes for N/N and N/A 2D gel analysis are shown. Expanded view of the 1.9 kb 5 NTS. Thick arrow- rRNA promoter; grey ovals- position of phase nucleosomes in vegetative rDNA minichromosomes [ 60 ], black boxes- type 1 repeats. Domains 1 and 2 (thin arrows) are 430 bp imperfect tandem repeats with 230 bp nuclease hypersensitive regions Lower diagram: schematic of typical RI patterns detected by N/N 2D gel electrophoresis. Simple Y arc (arrow): passive replication of the probed DNA fragment interval due to initiation elsewhere in the chromosome. Bubble arc (arrowhead): initiation at a central position in the probed DNA fragment. Bubble-to-Y arc: initiation at an asymmetric position in the examined fragment (low MW RIs: bubble arc (arrowhead), high MW RIs: Y arc). Composite: active (complete bubble arc, arrowhead) and passive (complete Y arc, arrow) replication within the probed DNA fragment. X and Double Y: the X spike (arrowhead) is generated from branch migration recombinant intermediates, and the double Y (open arrow) is generated by two converging replication forks. (B) RI patterns detected with the 5’ NTS probe 1 on HindIII digested DNA from mock treated quiescent (starvation) and replicating cell populations (S phase). (C) 5’ NTS analysis on DNA from HU-treated cells 0–120 min after HU removal (arrow: simple Y arc, passive replication; arrowhead: X-spike recombination intermediates). See flow cytometry profiles ( Fig 5A ) and western blots ( Fig 5B ) for cell cycle progression and abundance of Orc1p and Mcm6p, respectively. (D) Two-dimensional N-N gel analysis of the 5.5 kb rRNA coding region ClaI fragment (position 2168–7629, probe 2). (E) Two dimensional neutral-alkaline (N/A) analysis of RIs derived from the rRNA coding region ClaI fragment. Probe 3 spans nucleotides 2169 to 3670, and probe 4 spans nucleotides 5214–6676. Schematic of nascent-strand RIs resolved by N/A 2D gel electrophoresis. The 1n spot corresponds to non-replicating DNA. The vertical smear is derived from nicked, non-replicating DNA, and the horizontal smear represents the parental strand in RIs of different length. The diagonal arc corresponds to nascent-strand replication intermediates that are liberated from the parental strand by alkali denaturation prior to electrophoresis in the second dimension.

    Article Snippet: For RI enrichment, 200 μg of genomic DNA were digested with HindIII (rDNA 5’ NTS analysis) or ClaI (rDNA coding region analysis) for 4 h and applied to the 200-μl packed volume benzoylated naphthoylated DEAE (BND)-cellulose (Sigma-Aldrich).

    Techniques: Two-Dimensional Gel Electrophoresis, Electrophoresis, Cell Culture, Southern Blot, Generated, Migration, Recombinant, Flow Cytometry, Cytometry, Western Blot, Derivative Assay

    Modulation of miRNA levels by Cholesterol, DHA and CLA. Differentiated Caco-2 cells were treated with micelles containing cholesterol (250 μM), DHA (200 μM), CLA (200 μM) or nothing (empty micelles) for 24 (DHA and CLA) and 48

    Journal: Molecular nutrition & food research

    Article Title: Dietary lipids modulate the expression of miR-107, a miRNA that regulates the circadian system

    doi: 10.1002/mnfr.201400616

    Figure Lengend Snippet: Modulation of miRNA levels by Cholesterol, DHA and CLA. Differentiated Caco-2 cells were treated with micelles containing cholesterol (250 μM), DHA (200 μM), CLA (200 μM) or nothing (empty micelles) for 24 (DHA and CLA) and 48

    Article Snippet: Cholesterol (Chol), CLA and DHA (Sigma, Madrid, Spain) were delivered to cells as micelles with Lyso-phosphatidilcholine (Lyso-PC; Sigma, Madrid, Spain) and sodium taurocholate (Tau; Sigma, Madrid, Spain).

    Techniques:

    Organization of the PDE1 Locus and Targeted Gene Disruption. (A) Restriction map of the insertional mutant locus of pde1 ) are indicated ( HPH ). pCB1003 integration was found to have occurred 165 bp upstream of a large open reading frame (gray shading). ClaI sites found on either side of the integrated plasmid were used to excise a fragment of the insertion locus. (B) Restriction map of the PDE1 locus isolated as a 6.94-kb PstI-SalI fragment from a Magnaporthe strain Guy11 genomic library. The PDE1 locus contains a 4575-bp open reading frame interrupted by a single intron at positions 3927 to 3997. The arrow indicates the orientation of the open reading frame and the position of the intron. (C) Targeted gene disruption vector pPde1::Hph. The vector was made by excising a 2.6-kb PstI fragment at the 5′ end of PDE1 and inserting the hygromycin B resistance gene cassette ( HPH ) in a SmaI site. (D) Restriction map of the pde1 :: Hph disruption allele showing the position of the hygromycin resistance gene cassette ( HPH ) at the 5′ end of the PDE1 open reading frame. (E) DNA gel blot analysis of pPde1::Hph transformants. Genomic DNA was prepared from the wild-type strain Guy11 (lane 1), the ectopic integration transformant PV9 (lane 2), and four pde1 :: Hph mutant transformants, PV1, PV2, PV3, and PV4 (lanes 3 to 6, respectively). Genomic DNA was digested with PstI and separated on a 0.8% agarose gel. The blot was probed with pSKPS1. The Xs between (B) and (C) indicate a crossover event. B, BamHI; C, ClaI; P, PstI; Sa, SalI; Sm, SmaI; X, XhoI.

    Journal: The Plant Cell

    Article Title: PDE1 Encodes a P-Type ATPase Involved in Appressorium-Mediated Plant Infection by the Rice Blast Fungus Magnaporthe grisea

    doi: 10.1105/TPC.010056

    Figure Lengend Snippet: Organization of the PDE1 Locus and Targeted Gene Disruption. (A) Restriction map of the insertional mutant locus of pde1 ) are indicated ( HPH ). pCB1003 integration was found to have occurred 165 bp upstream of a large open reading frame (gray shading). ClaI sites found on either side of the integrated plasmid were used to excise a fragment of the insertion locus. (B) Restriction map of the PDE1 locus isolated as a 6.94-kb PstI-SalI fragment from a Magnaporthe strain Guy11 genomic library. The PDE1 locus contains a 4575-bp open reading frame interrupted by a single intron at positions 3927 to 3997. The arrow indicates the orientation of the open reading frame and the position of the intron. (C) Targeted gene disruption vector pPde1::Hph. The vector was made by excising a 2.6-kb PstI fragment at the 5′ end of PDE1 and inserting the hygromycin B resistance gene cassette ( HPH ) in a SmaI site. (D) Restriction map of the pde1 :: Hph disruption allele showing the position of the hygromycin resistance gene cassette ( HPH ) at the 5′ end of the PDE1 open reading frame. (E) DNA gel blot analysis of pPde1::Hph transformants. Genomic DNA was prepared from the wild-type strain Guy11 (lane 1), the ectopic integration transformant PV9 (lane 2), and four pde1 :: Hph mutant transformants, PV1, PV2, PV3, and PV4 (lanes 3 to 6, respectively). Genomic DNA was digested with PstI and separated on a 0.8% agarose gel. The blot was probed with pSKPS1. The Xs between (B) and (C) indicate a crossover event. B, BamHI; C, ClaI; P, PstI; Sa, SalI; Sm, SmaI; X, XhoI.

    Article Snippet: Restriction mapping revealed the presence of a ClaI site that was absent in the vector; therefore, 2029 genomic DNA was digested with ClaI and religated using T4 DNA ligase (Promega) at 14°C.

    Techniques: Mutagenesis, Plasmid Preparation, Isolation, Western Blot, Agarose Gel Electrophoresis

    Electropherograms of the T-RFs produced by Alu I- Bse RI (AB) digestion (A, C, E, G, I, and K) and Sma I- Cla I- Xba I (SCX) digestion (B, D, F, H, J, and L) of PCR amplicons from 16S rRNA genes from the reference lines of the pea aphid A. pisum LMB95/28 (A and B), R (C and D), IS (E and F), FH (G and H), and MD (I and J) and aphid 1330 from the natural population of A. pisum (K and L). The T-RFs are shown in black, and the internal standards (50, 75, 100, 139, 150, 160, 200, 250, 300, 340, 350, 400, 450, 490, and 500 bp) are shown in gray.

    Journal: Applied and Environmental Microbiology

    Article Title: Diversity of Bacteria Associated with Natural Aphid Populations

    doi: 10.1128/AEM.69.12.7216-7223.2003

    Figure Lengend Snippet: Electropherograms of the T-RFs produced by Alu I- Bse RI (AB) digestion (A, C, E, G, I, and K) and Sma I- Cla I- Xba I (SCX) digestion (B, D, F, H, J, and L) of PCR amplicons from 16S rRNA genes from the reference lines of the pea aphid A. pisum LMB95/28 (A and B), R (C and D), IS (E and F), FH (G and H), and MD (I and J) and aphid 1330 from the natural population of A. pisum (K and L). The T-RFs are shown in black, and the internal standards (50, 75, 100, 139, 150, 160, 200, 250, 300, 340, 350, 400, 450, 490, and 500 bp) are shown in gray.

    Article Snippet: A sample of the PCR product from each amplification was run on a 1.5% agarose gel, the remainder of the sample was purified with the Qiaquick PCR purification kit (Qiagen) as specified by the manufacturer, and subsamples (15 μl) were digested either with 3 U of Alu I and 2.5 U of Bse R1 (New England Biolabs) (37°C for 2 h) (AB digestion) or sequentially with 3 U of Sma I (25°C for 2 h) followed by 3 U of Cla I and 3 U of Xba I (37°C for 2 h) (Promega) (SCX digestion).

    Techniques: Produced, Polymerase Chain Reaction