new england biolabs kits  (New England Biolabs)


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    Structured Review

    New England Biolabs new england biolabs kits
    New England Biolabs Kits, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/new england biolabs kits/product/New England Biolabs
    Average 94 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    new england biolabs kits - by Bioz Stars, 2020-04
    94/100 stars

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    Related Articles

    Sequencing:

    Article Title: A Microbial Avenue to Cell Cycle Control in the Plant Superkingdom [C]A Microbial Avenue to Cell Cycle Control in the Plant Superkingdom [C] [W]A Microbial Avenue to Cell Cycle Control in the Plant Superkingdom [C] [W] [OPEN]
    Article Snippet: .. Illumina TruSeq sequencing adapters were ligated to dA-tailed DNA. (All enzymatic reactions were with New England Biolabs kits.) .. Adaptor-ligated DNA (300 to 600 bp) was purified (Qiagen).

    Article Title: Comprehensive Discovery of Cell-Cycle-Essential Pathways in Chlamydomonas reinhardtii
    Article Snippet: Illumina TruSeq sequencing adapters were ligated to dA-tailed DNA. .. All enzymatic reactions were performed using New England Biolabs kits.

    Article Title: Mast cells regulate CD4+ T-cell differentiation in the absence of antigen presentation
    Article Snippet: After 16 hours of culture, cells were collected and RNA was extracted with the RNAqueous extraction kit, according to the manufacturer’s protocols (Applied Biosystems), as described above. cDNAwas obtained by using New England Biolabs kits (NEBNext Ultra Directional RNA Library Prep Kit for Illumina; New England Biolabs, Ipswich, Mass). .. Library preparation was performed by ligating on the P5 and P7 Illumina adaptors along with an index and amplifying the sequencing library by using PCR.

    Sample Prep:

    Article Title: Comprehensive Discovery of Cell-Cycle-Essential Pathways in Chlamydomonas reinhardtii
    Article Snippet: Paragraph title: Genomic Sample Preparation ... All enzymatic reactions were performed using New England Biolabs kits.

    Sonication:

    Article Title: A Microbial Avenue to Cell Cycle Control in the Plant Superkingdom [C]A Microbial Avenue to Cell Cycle Control in the Plant Superkingdom [C] [W]A Microbial Avenue to Cell Cycle Control in the Plant Superkingdom [C] [W] [OPEN]
    Article Snippet: Five micrograms of sonicated DNA was end-repaired and dA-tailed. .. Illumina TruSeq sequencing adapters were ligated to dA-tailed DNA. (All enzymatic reactions were with New England Biolabs kits.)

    Article Title: Comprehensive Discovery of Cell-Cycle-Essential Pathways in Chlamydomonas reinhardtii
    Article Snippet: Samples were treated with RNase A, and purified DNA was fragmented by sonication (Covaris S2 Focused Ultrasonicator) to 300 bp, followed by gel excision of a size range from 200 to 500 bp. .. All enzymatic reactions were performed using New England Biolabs kits.

    Magnetic Beads:

    Article Title: Mast cells regulate CD4+ T-cell differentiation in the absence of antigen presentation
    Article Snippet: After 16 hours of culture, cells were collected and RNA was extracted with the RNAqueous extraction kit, according to the manufacturer’s protocols (Applied Biosystems), as described above. cDNAwas obtained by using New England Biolabs kits (NEBNext Ultra Directional RNA Library Prep Kit for Illumina; New England Biolabs, Ipswich, Mass). .. Briefly, mRNAwas extracted with polyT magnetic beads, and then first- and second-strand syntheses were performed.

    Ligation:

    Article Title: Comprehensive Discovery of Cell-Cycle-Essential Pathways in Chlamydomonas reinhardtii
    Article Snippet: All enzymatic reactions were performed using New England Biolabs kits. .. Efficiency of end modification is tested as follows: (1) In a small test ligation, sonicated DNA self-ligates very inefficiently (most ends staggered).

    RNA Sequencing Assay:

    Article Title: Mast cells regulate CD4+ T-cell differentiation in the absence of antigen presentation
    Article Snippet: Paragraph title: RNA sequencing analysis ... After 16 hours of culture, cells were collected and RNA was extracted with the RNAqueous extraction kit, according to the manufacturer’s protocols (Applied Biosystems), as described above. cDNAwas obtained by using New England Biolabs kits (NEBNext Ultra Directional RNA Library Prep Kit for Illumina; New England Biolabs, Ipswich, Mass).

    Cell Culture:

    Article Title: Mast cells regulate CD4+ T-cell differentiation in the absence of antigen presentation
    Article Snippet: BMMCs from C57BL/6 mice were cultured in the presence of NAD+ (500 µmol/L), LPS (10 µg/mL; Escherichia coli O127:B8), or placebo (PBS). .. After 16 hours of culture, cells were collected and RNA was extracted with the RNAqueous extraction kit, according to the manufacturer’s protocols (Applied Biosystems), as described above. cDNAwas obtained by using New England Biolabs kits (NEBNext Ultra Directional RNA Library Prep Kit for Illumina; New England Biolabs, Ipswich, Mass).

    Purification:

    Article Title: A Microbial Avenue to Cell Cycle Control in the Plant Superkingdom [C]A Microbial Avenue to Cell Cycle Control in the Plant Superkingdom [C] [W]A Microbial Avenue to Cell Cycle Control in the Plant Superkingdom [C] [W] [OPEN]
    Article Snippet: Ten micrograms of RNase A was added, 37°C for 1 h, and DNA purified with the Qiaex gel extraction kit (Qiagen). .. Illumina TruSeq sequencing adapters were ligated to dA-tailed DNA. (All enzymatic reactions were with New England Biolabs kits.)

    Article Title: Comprehensive Discovery of Cell-Cycle-Essential Pathways in Chlamydomonas reinhardtii
    Article Snippet: Samples were treated with RNase A, and purified DNA was fragmented by sonication (Covaris S2 Focused Ultrasonicator) to 300 bp, followed by gel excision of a size range from 200 to 500 bp. .. All enzymatic reactions were performed using New England Biolabs kits.

    Real-time Polymerase Chain Reaction:

    Article Title: Comprehensive Discovery of Cell-Cycle-Essential Pathways in Chlamydomonas reinhardtii
    Article Snippet: All enzymatic reactions were performed using New England Biolabs kits. .. DNA concentration was estimated both by agarose gel electrophoresis and conventional qPCR using seven dilutions (500×–32,000× in 2× dilution steps) of a standard sample.

    Concentration Assay:

    Article Title: Comprehensive Discovery of Cell-Cycle-Essential Pathways in Chlamydomonas reinhardtii
    Article Snippet: All enzymatic reactions were performed using New England Biolabs kits. .. DNA concentration was estimated both by agarose gel electrophoresis and conventional qPCR using seven dilutions (500×–32,000× in 2× dilution steps) of a standard sample.

    Generated:

    Article Title: Mast cells regulate CD4+ T-cell differentiation in the absence of antigen presentation
    Article Snippet: After 16 hours of culture, cells were collected and RNA was extracted with the RNAqueous extraction kit, according to the manufacturer’s protocols (Applied Biosystems), as described above. cDNAwas obtained by using New England Biolabs kits (NEBNext Ultra Directional RNA Library Prep Kit for Illumina; New England Biolabs, Ipswich, Mass). .. Once double-strand cDNA was generated, DNA was cleaned up with magnetic beads and then went into library prep.

    Gel Extraction:

    Article Title: A Microbial Avenue to Cell Cycle Control in the Plant Superkingdom [C]A Microbial Avenue to Cell Cycle Control in the Plant Superkingdom [C] [W]A Microbial Avenue to Cell Cycle Control in the Plant Superkingdom [C] [W] [OPEN]
    Article Snippet: Ten micrograms of RNase A was added, 37°C for 1 h, and DNA purified with the Qiaex gel extraction kit (Qiagen). .. Illumina TruSeq sequencing adapters were ligated to dA-tailed DNA. (All enzymatic reactions were with New England Biolabs kits.)

    Article Title: Comprehensive Discovery of Cell-Cycle-Essential Pathways in Chlamydomonas reinhardtii
    Article Snippet: Following gel extraction, DNA was end-repaired and dA-tailed. .. All enzymatic reactions were performed using New England Biolabs kits.

    Modification:

    Article Title: Comprehensive Discovery of Cell-Cycle-Essential Pathways in Chlamydomonas reinhardtii
    Article Snippet: All enzymatic reactions were performed using New England Biolabs kits. .. Efficiency of end modification is tested as follows: (1) In a small test ligation, sonicated DNA self-ligates very inefficiently (most ends staggered).

    Agarose Gel Electrophoresis:

    Article Title: Comprehensive Discovery of Cell-Cycle-Essential Pathways in Chlamydomonas reinhardtii
    Article Snippet: All enzymatic reactions were performed using New England Biolabs kits. .. DNA concentration was estimated both by agarose gel electrophoresis and conventional qPCR using seven dilutions (500×–32,000× in 2× dilution steps) of a standard sample.

    Mouse Assay:

    Article Title: Mast cells regulate CD4+ T-cell differentiation in the absence of antigen presentation
    Article Snippet: BMMCs from C57BL/6 mice were cultured in the presence of NAD+ (500 µmol/L), LPS (10 µg/mL; Escherichia coli O127:B8), or placebo (PBS). .. After 16 hours of culture, cells were collected and RNA was extracted with the RNAqueous extraction kit, according to the manufacturer’s protocols (Applied Biosystems), as described above. cDNAwas obtained by using New England Biolabs kits (NEBNext Ultra Directional RNA Library Prep Kit for Illumina; New England Biolabs, Ipswich, Mass).

    Polymerase Chain Reaction:

    Article Title: Mast cells regulate CD4+ T-cell differentiation in the absence of antigen presentation
    Article Snippet: After 16 hours of culture, cells were collected and RNA was extracted with the RNAqueous extraction kit, according to the manufacturer’s protocols (Applied Biosystems), as described above. cDNAwas obtained by using New England Biolabs kits (NEBNext Ultra Directional RNA Library Prep Kit for Illumina; New England Biolabs, Ipswich, Mass). .. Library preparation was performed by ligating on the P5 and P7 Illumina adaptors along with an index and amplifying the sequencing library by using PCR.

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    Removal of excess primers and unincorporated dNTPs from PCR products is recommended prior to downstream applications and analysis The Exo CIP Rapid PCR Cleanup Kit serves this purpose by utilizing
      Buy from Supplier

    93
    New England Biolabs cdna synthesis kit
    Confirmation of pET28 (a) ProB and pET22 (b) ProB clones with <t>PCR</t> and double digestion. ( A ) Lane 1 2 represents PCR amplified 639 bp fragment using pET28 (a) ProB construct as template. M, low range DNA ruler (Bangalore Genei). Lane 3 4 represents PCR amplified 639 bp fragment using pET22 (b) ProB construct as template. ( B ) . Lane 1 represents pET28 (a) ProB construct digested with Nhe1. The DNA fragment of 5974 bp represents the combined size of pET-28a(+) and Procerain B <t>cDNA.</t> Lane M represents 1 kb DNA ladder (NEB). Lane 2 represents release of 639 bp fragment (Procerain B cDNA) on double digestion (Nhe1/BamH1) of pET28 (a) ProB construct. ( C ) . Lane 1 represents release of 639 bp fragment (Procerain B cDNA) on double digestion (BamH1/Xho1) of pET22 (b) ProB construct. Lane M represents 1 kb DNA ladder (NEB). Lane 2 represents pET22 (b) ProB construct digested with BamH1. The DNA fragment of 6092 bp represents the combined size of pET-22b(+) and Procerain B cDNA. ( D ) SDS-PAGE showing the over expression of recombinant procerain B in BL21 transformed with pET28 (a) ProB. Lane M represents medium range protein molecular weight marker (Bangalore Genei). Lane 1 and 2 represent supernatant and pellet of induced culture (1 mM IPTG) at 37°C. Lane 3 and 4 represent supernatant and pellet of un-induced culture at 37°C. Lane 5 6 represents supernatant and pellet of induced (1 mM IPTG) at 25°C. Lane 7 and 8 represent supernatant and pellet of uninduced culture at 25°C. Lane 9 represents pellet of BL21 transformed with pET28a(+) at 25°C. ( E ) . Expression profile of recombinant procerain B in BL21 transformed with pET22 (b) ProB. Lane 1 and 2 represent pellet and supernatant of induced culture (1 mM IPTG) at 25°C. M . Medium range protein molecular weight marker (Bangalore Genei). Lane 3 and 4 represent supernatant and pellet of un-induced culture at 20°C. ( F ) . Comparison of native and recombinant procerain B (with His-tag) on SDS-PAGE. Lane 1 represents purified native procerain B. Lane M represents medium range protein molecular weight marker (Bangalore Genei). Lane 2 represents purified recombinant procerain B (with His-tag). Slight difference in molecular weight of native and recombinant procerain B is due to presence of His-tag in recombinant procerain B. Arrow in Figure 3 (D, E, F) indicates recombinant procerain B.
    Cdna Synthesis Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cdna synthesis kit/product/New England Biolabs
    Average 93 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    cdna synthesis kit - by Bioz Stars, 2020-04
    93/100 stars
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    86
    New England Biolabs ipec j2
    Analysis of CAV2-miR-29a interaction in humans and pigs by means of reporter gene assays. The interaction between miR-29a and human as well as porcine CAV2 was verified by means of reporter gene assays using HeLa and <t>IPEC-J2</t> cells, respectively. Identified target sites between miR-29a and human (panel A) as well as porcine CAV2 (panel B) were analysed using RNAhybrid. Relative luciferase activity (Luc Gaussia : Luc Cypridina ) was determined respective to nonsense miRNA mimics as well as mutagenised seeds (red letters) serving as controls. The columns show means of normalised luciferase activity each measured in triplicates while error bars show the standard deviation. Asterisks indicate statistical significance between samples (*: P
    Ipec J2, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 86 stars, based on 1 article reviews
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    91
    New England Biolabs srna sequencing data
    Gel mobility shift assays showing preferential RNA binding of recombinant PPR103 (rPPR103) to rpl16 <t>sRNA.</t> ( A ) Purification of rPPR103. 100 and 200 ng of purified rPPR103 and MBP, respectively, were analyzed by SDS-PAGE and staining with Coomassie Brilliant Blue. The predicted sizes of rPPR103 and MBP are 129 and 44 kDa, respectively. ( B ) Gel mobility shift assays with rPPR103. The <t>RNAs</t> used in the binding assays were gel purified and ∼100 ng of each was resolved on a 12% denaturing polyacrylamide gel and stained with ethidium bromide to assess their purity (left panel), and 30 pM of radiolabeled RNAs were incubated with increasing concentrations of rPPR103 (0, 7.5, 15, 30 nM) or MBP (30 nM). The binding assays were run on a native acrylamide gel. Bound (B) and unbound (U) RNAs are indicated. The sequences of the RNAs are shown below. ( C ) Gel mobility shift assays using unlabeled RNA competitors. The rPPR103 concentration was kept constant (30 nM) and the molar excess of cold RNAs relative to the labeled rpl16 RNA (30 pM) is indicated at the top of the gel. Quantification of the amount of radioactive RNA in the bound fraction is shown to the right.
    Srna Sequencing Data, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 91 stars, based on 1 article reviews
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    Image Search Results


    Confirmation of pET28 (a) ProB and pET22 (b) ProB clones with PCR and double digestion. ( A ) Lane 1 2 represents PCR amplified 639 bp fragment using pET28 (a) ProB construct as template. M, low range DNA ruler (Bangalore Genei). Lane 3 4 represents PCR amplified 639 bp fragment using pET22 (b) ProB construct as template. ( B ) . Lane 1 represents pET28 (a) ProB construct digested with Nhe1. The DNA fragment of 5974 bp represents the combined size of pET-28a(+) and Procerain B cDNA. Lane M represents 1 kb DNA ladder (NEB). Lane 2 represents release of 639 bp fragment (Procerain B cDNA) on double digestion (Nhe1/BamH1) of pET28 (a) ProB construct. ( C ) . Lane 1 represents release of 639 bp fragment (Procerain B cDNA) on double digestion (BamH1/Xho1) of pET22 (b) ProB construct. Lane M represents 1 kb DNA ladder (NEB). Lane 2 represents pET22 (b) ProB construct digested with BamH1. The DNA fragment of 6092 bp represents the combined size of pET-22b(+) and Procerain B cDNA. ( D ) SDS-PAGE showing the over expression of recombinant procerain B in BL21 transformed with pET28 (a) ProB. Lane M represents medium range protein molecular weight marker (Bangalore Genei). Lane 1 and 2 represent supernatant and pellet of induced culture (1 mM IPTG) at 37°C. Lane 3 and 4 represent supernatant and pellet of un-induced culture at 37°C. Lane 5 6 represents supernatant and pellet of induced (1 mM IPTG) at 25°C. Lane 7 and 8 represent supernatant and pellet of uninduced culture at 25°C. Lane 9 represents pellet of BL21 transformed with pET28a(+) at 25°C. ( E ) . Expression profile of recombinant procerain B in BL21 transformed with pET22 (b) ProB. Lane 1 and 2 represent pellet and supernatant of induced culture (1 mM IPTG) at 25°C. M . Medium range protein molecular weight marker (Bangalore Genei). Lane 3 and 4 represent supernatant and pellet of un-induced culture at 20°C. ( F ) . Comparison of native and recombinant procerain B (with His-tag) on SDS-PAGE. Lane 1 represents purified native procerain B. Lane M represents medium range protein molecular weight marker (Bangalore Genei). Lane 2 represents purified recombinant procerain B (with His-tag). Slight difference in molecular weight of native and recombinant procerain B is due to presence of His-tag in recombinant procerain B. Arrow in Figure 3 (D, E, F) indicates recombinant procerain B.

    Journal: PLoS ONE

    Article Title: cDNA Cloning and Molecular Modeling of Procerain B, a Novel Cysteine Endopeptidase Isolated from Calotropis procera

    doi: 10.1371/journal.pone.0059806

    Figure Lengend Snippet: Confirmation of pET28 (a) ProB and pET22 (b) ProB clones with PCR and double digestion. ( A ) Lane 1 2 represents PCR amplified 639 bp fragment using pET28 (a) ProB construct as template. M, low range DNA ruler (Bangalore Genei). Lane 3 4 represents PCR amplified 639 bp fragment using pET22 (b) ProB construct as template. ( B ) . Lane 1 represents pET28 (a) ProB construct digested with Nhe1. The DNA fragment of 5974 bp represents the combined size of pET-28a(+) and Procerain B cDNA. Lane M represents 1 kb DNA ladder (NEB). Lane 2 represents release of 639 bp fragment (Procerain B cDNA) on double digestion (Nhe1/BamH1) of pET28 (a) ProB construct. ( C ) . Lane 1 represents release of 639 bp fragment (Procerain B cDNA) on double digestion (BamH1/Xho1) of pET22 (b) ProB construct. Lane M represents 1 kb DNA ladder (NEB). Lane 2 represents pET22 (b) ProB construct digested with BamH1. The DNA fragment of 6092 bp represents the combined size of pET-22b(+) and Procerain B cDNA. ( D ) SDS-PAGE showing the over expression of recombinant procerain B in BL21 transformed with pET28 (a) ProB. Lane M represents medium range protein molecular weight marker (Bangalore Genei). Lane 1 and 2 represent supernatant and pellet of induced culture (1 mM IPTG) at 37°C. Lane 3 and 4 represent supernatant and pellet of un-induced culture at 37°C. Lane 5 6 represents supernatant and pellet of induced (1 mM IPTG) at 25°C. Lane 7 and 8 represent supernatant and pellet of uninduced culture at 25°C. Lane 9 represents pellet of BL21 transformed with pET28a(+) at 25°C. ( E ) . Expression profile of recombinant procerain B in BL21 transformed with pET22 (b) ProB. Lane 1 and 2 represent pellet and supernatant of induced culture (1 mM IPTG) at 25°C. M . Medium range protein molecular weight marker (Bangalore Genei). Lane 3 and 4 represent supernatant and pellet of un-induced culture at 20°C. ( F ) . Comparison of native and recombinant procerain B (with His-tag) on SDS-PAGE. Lane 1 represents purified native procerain B. Lane M represents medium range protein molecular weight marker (Bangalore Genei). Lane 2 represents purified recombinant procerain B (with His-tag). Slight difference in molecular weight of native and recombinant procerain B is due to presence of His-tag in recombinant procerain B. Arrow in Figure 3 (D, E, F) indicates recombinant procerain B.

    Article Snippet: PCR master mix, cDNA synthesis kit, NheI, BamHI, XhoI restriction endonucleases and T4 DNA ligase were purchased from New England Biolabs (NEB, England).

    Techniques: Clone Assay, Polymerase Chain Reaction, Amplification, Construct, Positron Emission Tomography, SDS Page, Over Expression, Recombinant, Transformation Assay, Molecular Weight, Marker, Expressing, Purification

    Analysis of CAV2-miR-29a interaction in humans and pigs by means of reporter gene assays. The interaction between miR-29a and human as well as porcine CAV2 was verified by means of reporter gene assays using HeLa and IPEC-J2 cells, respectively. Identified target sites between miR-29a and human (panel A) as well as porcine CAV2 (panel B) were analysed using RNAhybrid. Relative luciferase activity (Luc Gaussia : Luc Cypridina ) was determined respective to nonsense miRNA mimics as well as mutagenised seeds (red letters) serving as controls. The columns show means of normalised luciferase activity each measured in triplicates while error bars show the standard deviation. Asterisks indicate statistical significance between samples (*: P

    Journal: PLoS ONE

    Article Title: Intestinal Salmonella typhimurium Infection Leads to miR-29a Induced Caveolin 2 Regulation

    doi: 10.1371/journal.pone.0067300

    Figure Lengend Snippet: Analysis of CAV2-miR-29a interaction in humans and pigs by means of reporter gene assays. The interaction between miR-29a and human as well as porcine CAV2 was verified by means of reporter gene assays using HeLa and IPEC-J2 cells, respectively. Identified target sites between miR-29a and human (panel A) as well as porcine CAV2 (panel B) were analysed using RNAhybrid. Relative luciferase activity (Luc Gaussia : Luc Cypridina ) was determined respective to nonsense miRNA mimics as well as mutagenised seeds (red letters) serving as controls. The columns show means of normalised luciferase activity each measured in triplicates while error bars show the standard deviation. Asterisks indicate statistical significance between samples (*: P

    Article Snippet: Nucleofection was performed using 5×105 –1×106 of either HeLa (Kit R) or HT-29 (Kit R) or IPEC-J2 (Kit L) using 1–2 µg reporter plasmid (pTK-Gluc derivatives, NEB GmbH), 100–200 ng normalisation plasmid (pTK-Cluc, NEB GmbH) and 100 pmol miRNA mimic according to the manufacturer’s instructions.

    Techniques: Luciferase, Activity Assay, Standard Deviation

    Immuno detection of CAV2 and CDC42 after RNAi and overexpression experiments in HT-29 and IPEC-J2 intestinal cells. Panel A exemplifies the expression of CAV2 as well as CDC42 after RNAi (nonsense controls, anti-miR-29a, miR-29a and siRNA CAV2) and transient overexpression of CAV2 (pMIREX0 as a mock control and pMIREXCAV2) in HT-29 cells. Panel B shows immunocyto detection of CAV2 and CDC42 after transfection (according to panel A) in intestinal cell lines of both investigated species human (HT-29) and pig (IPEC-J2). Scales are indicated by white bars (10 µm). Overview images are included in the bottom right corner.

    Journal: PLoS ONE

    Article Title: Intestinal Salmonella typhimurium Infection Leads to miR-29a Induced Caveolin 2 Regulation

    doi: 10.1371/journal.pone.0067300

    Figure Lengend Snippet: Immuno detection of CAV2 and CDC42 after RNAi and overexpression experiments in HT-29 and IPEC-J2 intestinal cells. Panel A exemplifies the expression of CAV2 as well as CDC42 after RNAi (nonsense controls, anti-miR-29a, miR-29a and siRNA CAV2) and transient overexpression of CAV2 (pMIREX0 as a mock control and pMIREXCAV2) in HT-29 cells. Panel B shows immunocyto detection of CAV2 and CDC42 after transfection (according to panel A) in intestinal cell lines of both investigated species human (HT-29) and pig (IPEC-J2). Scales are indicated by white bars (10 µm). Overview images are included in the bottom right corner.

    Article Snippet: Nucleofection was performed using 5×105 –1×106 of either HeLa (Kit R) or HT-29 (Kit R) or IPEC-J2 (Kit L) using 1–2 µg reporter plasmid (pTK-Gluc derivatives, NEB GmbH), 100–200 ng normalisation plasmid (pTK-Cluc, NEB GmbH) and 100 pmol miRNA mimic according to the manufacturer’s instructions.

    Techniques: Over Expression, Expressing, Transfection

    Gel mobility shift assays showing preferential RNA binding of recombinant PPR103 (rPPR103) to rpl16 sRNA. ( A ) Purification of rPPR103. 100 and 200 ng of purified rPPR103 and MBP, respectively, were analyzed by SDS-PAGE and staining with Coomassie Brilliant Blue. The predicted sizes of rPPR103 and MBP are 129 and 44 kDa, respectively. ( B ) Gel mobility shift assays with rPPR103. The RNAs used in the binding assays were gel purified and ∼100 ng of each was resolved on a 12% denaturing polyacrylamide gel and stained with ethidium bromide to assess their purity (left panel), and 30 pM of radiolabeled RNAs were incubated with increasing concentrations of rPPR103 (0, 7.5, 15, 30 nM) or MBP (30 nM). The binding assays were run on a native acrylamide gel. Bound (B) and unbound (U) RNAs are indicated. The sequences of the RNAs are shown below. ( C ) Gel mobility shift assays using unlabeled RNA competitors. The rPPR103 concentration was kept constant (30 nM) and the molar excess of cold RNAs relative to the labeled rpl16 RNA (30 pM) is indicated at the top of the gel. Quantification of the amount of radioactive RNA in the bound fraction is shown to the right.

    Journal: Nucleic Acids Research

    Article Title: A PPR protein in the PLS subfamily stabilizes the 5′-end of processed rpl16 mRNAs in maize chloroplasts

    doi: 10.1093/nar/gkw270

    Figure Lengend Snippet: Gel mobility shift assays showing preferential RNA binding of recombinant PPR103 (rPPR103) to rpl16 sRNA. ( A ) Purification of rPPR103. 100 and 200 ng of purified rPPR103 and MBP, respectively, were analyzed by SDS-PAGE and staining with Coomassie Brilliant Blue. The predicted sizes of rPPR103 and MBP are 129 and 44 kDa, respectively. ( B ) Gel mobility shift assays with rPPR103. The RNAs used in the binding assays were gel purified and ∼100 ng of each was resolved on a 12% denaturing polyacrylamide gel and stained with ethidium bromide to assess their purity (left panel), and 30 pM of radiolabeled RNAs were incubated with increasing concentrations of rPPR103 (0, 7.5, 15, 30 nM) or MBP (30 nM). The binding assays were run on a native acrylamide gel. Bound (B) and unbound (U) RNAs are indicated. The sequences of the RNAs are shown below. ( C ) Gel mobility shift assays using unlabeled RNA competitors. The rPPR103 concentration was kept constant (30 nM) and the molar excess of cold RNAs relative to the labeled rpl16 RNA (30 pM) is indicated at the top of the gel. Quantification of the amount of radioactive RNA in the bound fraction is shown to the right.

    Article Snippet: The sRNA sequencing data were obtained by gel-purifying RNAs between ∼15 and 40 nts from maize seedling leaf RNA, generating sequencing libraries with the NEBNext Multiplex Small RNA Library Prep Set, and sequencing on an Illumina HiSeq2000 at the University of Oregon Genomics Core Facility.

    Techniques: Mobility Shift, RNA Binding Assay, Recombinant, Purification, SDS Page, Staining, Binding Assay, Incubation, Acrylamide Gel Assay, Concentration Assay, Labeling