low molecular weight rna marker Search Results


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  • 99
    New England Biolabs range ssrna ladder
    Purification and in vitro cleavage assay of Synechocystis WT Cas6-1 and mutated Cas6-1 proteins. (A) Coomassie-stained 15% SDS-PAGE of heterologously overexpressed and purified Cas6-1 protein variants. The expected molecular weight of Cas6-1_6xHis is ∼ 30 kDa. 66.67 pmol recombinant enzyme was loaded on each lane and PageRuler™ Prestained Protein Ladder (Thermo Fisher Scientific) served for protein size estimation. (B, C) In in vitro cleavage assays both the (B) CRISPR1 RNA oligo repeat sequence and the (C) pre-acrRNA cassette is processed with variable efficiency by the purified Cas6-1 protein variants shown in (A). After incubation at 30°C the RNA fragments were electrophoretically size separated on a 15% PAA sequencing gel and visualized with SYBR® Gold Nucleic Acid Gel Stain. The processing results in a pattern of RNA fragments of distinct lengths. Cleavage assays were conducted with a three-fold excess (B) or a 1:1 ratio (C) of protein to RNA-substrate in cleavage buffer and incubated at 30°C for the indicated time. Expected RNA fragments and their corresponding sizes are illustrated by cartoons. Another Cas6-1 WT protein sample, which was overexpressed and purified independently, is indicated by an asterisk. Negative controls were complemented with 1 μL of H 2 O or 1 μL eluate of a mock control. Two additional nucleotides resulting from in vitro transcription were added to the 215 nt long acrRNA yielding a 217 nt long pre-acrRNA. For RNA size estimation a CRISPR1 repeat oligo ladder (C1L), <t>RiboRuler</t> low range RNA ladder (Thermo Fisher Scientific), low range <t>ssRNA</t> ladder or microRNA marker (both from New England Biolabs) were used.
    Range Ssrna Ladder, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 124 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher o generuler ultra low range dna ladder
    Electrophoretic analysis of partial pep A gene (596 bp) of B. pseudomallei digested with Stu I and Hinc II restriction analysis. (12% polyacrylamide gel, 1X TBE buffer, 8 V/cm, 130 min); Lane M- <t>O’GeneRuler™</t> ultra low range <t>DNA</t> ladder; Lane 1- B. pseudomallei NCTC 13178; Lane 2- B. pseudomallei ATCC 23343; Lane 3- Type I; Lane 4- Type II; Lane 5- Type III.
    O Generuler Ultra Low Range Dna Ladder, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/o generuler ultra low range dna ladder/product/Thermo Fisher
    Average 99 stars, based on 15 article reviews
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    94
    Thermo Fisher low molecular weight rna marker
    In vivo expression and SL trans -splicing of a chimeric TnI/β-gal mRNA from a TnI/β-gal gene construct lacking the SL sequence. ( A ) Expression of β-gal, revealed by X-Gal staining (blue), in tail muscle of embryos 12 h following introduction of CiTnILacZ(−1.5) DNA into zygotes by electroporation. ( B ) RT–PCR amplification of β-gal mRNA with β-gal-specific leftward priming, and rightward priming with the SL primer. (Lane 1 ) Size markers (MBI <t>Fermentas</t> ladder mix, 10–0.1 kb; top visible band, 3 kb, bottom visible band 500 bp). (Lanes 2,3 ) RT–PCR products from two different batches of transfected embryos. The template in lane 4 was tRNA, used as a carrier in embryo <t>RNA</t> isolations. A 550-bp product, the size predicted for SL-ended β-gal mRNA, was produced from both embryo batches. [Production of this product required the presence of both primers and did not occur when CiTnILacZ(−1.5) plasmid DNA was used as the amplification template. An additional product of ∼400 bp seen in lanes 2 and 3 required only the β-gal-specific primer and was produced in control amplifications of CiTnILacZ(−1.5) plasmid DNA; it apparently results from rightward mis-priming by the β-gal-specific primer upstream of its normal leftward priming site.] ( C ) DNA sequence of 550-bp RT–PCR product. The 550-bp product (as in B ) was recovered and sequenced using the SL primer ( right ) and β-gal-specific primer ( left ). The leftward sequence confirmed the presence of the SL primer (bold) immediately upstream of TnI mRNA nucleotide 17. Not shown is the Bam HI site engineered at the 5′-end of the SL primer. Sequences deriving from the β-gal reporter gene are shown in lower case.
    Low Molecular Weight Rna Marker, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/low molecular weight rna marker/product/Thermo Fisher
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    90
    Thermo Fisher low range rna molecular weight markers
    Genomic organization and expression strategy of Or-CD15 . ( A ) Screen shot of the RpL23A / Uhg7 region taken from the UCSC genome browser. The RpL23A-RA and Uhg7 transcripts are depicted in blue, the location of Or-CD15 and the newly identified transcript RpL23A-RB are shown in black. Below, sequence conservation as measured by phastcons and the coverage of a multiple genome alignment is indicated; ( B ) Northern blot analysis of adult fruit fly <t>RNA.</t> The genomic position of the utilised probe is outlined in ( A ). The band at about 100 nt corresponds to snoRNA Or-CD15 ; sizes are indicated by an RNA molecular weight ladder (RiboRuler Low Range, Life Technologies); ( C ) <t>PCR</t> experiments using primers annealing to RpL23A exon 1 and Uhg7 exon 4; amplification of the genomic DNA (gDNA) produces the expected fragment of about 2 kb; the same primers used in RT-PCR experiments successfully amplified a fragment of 1.2 kb, representative of the new RpL23A-RB transcript. In this transcript, RpL23A and Uhg7 sequences are fused to each other. On the right, a DNA molecular weight ladder (100bp DNA ladder, New England BioLabs, Ipswich, MA, USA) is given.
    Low Range Rna Molecular Weight Markers, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Thermo Fisher 100 nt rna low molecular weight marker
    Genomic organization and expression strategy of Or-CD15 . ( A ) Screen shot of the RpL23A / Uhg7 region taken from the UCSC genome browser. The RpL23A-RA and Uhg7 transcripts are depicted in blue, the location of Or-CD15 and the newly identified transcript RpL23A-RB are shown in black. Below, sequence conservation as measured by phastcons and the coverage of a multiple genome alignment is indicated; ( B ) Northern blot analysis of adult fruit fly <t>RNA.</t> The genomic position of the utilised probe is outlined in ( A ). The band at about 100 nt corresponds to snoRNA Or-CD15 ; sizes are indicated by an RNA molecular weight ladder (RiboRuler Low Range, Life Technologies); ( C ) <t>PCR</t> experiments using primers annealing to RpL23A exon 1 and Uhg7 exon 4; amplification of the genomic DNA (gDNA) produces the expected fragment of about 2 kb; the same primers used in RT-PCR experiments successfully amplified a fragment of 1.2 kb, representative of the new RpL23A-RB transcript. In this transcript, RpL23A and Uhg7 sequences are fused to each other. On the right, a DNA molecular weight ladder (100bp DNA ladder, New England BioLabs, Ipswich, MA, USA) is given.
    100 Nt Rna Low Molecular Weight Marker, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher riboruler low range rna ladder
    Incubation of in vitro transcripts of CRISPR1, 2 and 3 with Cas6-1. ( A ) A total of 2.3 μM of CRISPR1 transcript is cleaved by 6.2 μM Cas6-1 by incubation for 1 h. ( B ) A total of 2 μM of CRISPR2 transcript is cleaved by 6.2 μM Cas6-1 by incubation for 1 h. ( C ) A total of 3.2 μM of CRISPR3 transcript is not cleaved by 6.5 μM Cas6-1 by incubation for 2 h. ( D ) The cleavage of 6.6 μM of the shorter CRISPR1* in vitro transcript by 5.8 μM Cas6-1 monitored over a time of 2 h 20 min. Since no finally mature crRNA ( 17 ) was detected, the in vivo presence of an unknown ribonuclease is suggested. All reactions were performed at 37°C in a reaction volume of 5 μl. <t>RNA</t> was separated by 8 M urea 10% PAGE and bands were visualized by ethidium bromide staining. Diamonds represent repeats and rectangles spacer sequences. Transcripts were not gel purified, explaining the appearance of fragments smaller than the full length transcripts of CRISPR1 and 2 in absence of Cas6-1. Eb1, elution buffer 1 used as negative control. Molecular markers: MN, Low Range ssRNA Ladder (NEB); MF, <t>RiboRuler</t> Low Range RNA Ladder (Thermo Fisher Scientific).
    Riboruler Low Range Rna Ladder, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 99 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/riboruler low range rna ladder/product/Thermo Fisher
    Average 99 stars, based on 99 article reviews
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    99
    Thermo Fisher generuler ultra low range dna ladder
    Incubation of in vitro transcripts of CRISPR1, 2 and 3 with Cas6-1. ( A ) A total of 2.3 μM of CRISPR1 transcript is cleaved by 6.2 μM Cas6-1 by incubation for 1 h. ( B ) A total of 2 μM of CRISPR2 transcript is cleaved by 6.2 μM Cas6-1 by incubation for 1 h. ( C ) A total of 3.2 μM of CRISPR3 transcript is not cleaved by 6.5 μM Cas6-1 by incubation for 2 h. ( D ) The cleavage of 6.6 μM of the shorter CRISPR1* in vitro transcript by 5.8 μM Cas6-1 monitored over a time of 2 h 20 min. Since no finally mature crRNA ( 17 ) was detected, the in vivo presence of an unknown ribonuclease is suggested. All reactions were performed at 37°C in a reaction volume of 5 μl. <t>RNA</t> was separated by 8 M urea 10% PAGE and bands were visualized by ethidium bromide staining. Diamonds represent repeats and rectangles spacer sequences. Transcripts were not gel purified, explaining the appearance of fragments smaller than the full length transcripts of CRISPR1 and 2 in absence of Cas6-1. Eb1, elution buffer 1 used as negative control. Molecular markers: MN, Low Range ssRNA Ladder (NEB); MF, <t>RiboRuler</t> Low Range RNA Ladder (Thermo Fisher Scientific).
    Generuler Ultra Low Range Dna Ladder, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 134 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/generuler ultra low range dna ladder/product/Thermo Fisher
    Average 99 stars, based on 134 article reviews
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    99
    Millipore ethidium bromide stained 2
    Amplification of selected target gene fragments from DNA extracted from in situ dark- or light-adapted mature pin oak ( Q. palustris ) leaves. The figure shows a representative example of PCR amplification of the 1300 bp rbcL , 500 bp STS_8561_ palustris and 209 bp STS_8461_ palustris sequence tagged site segments from dark- (D) and light-adapted (L) leaves, using extraction method M1 and M2, on a 2% agarose gel containing 0.5 µg/mL <t>ethidium</t> bromide. The low molecular weight bands (at about 175 bp) in the lane showing the amplified rbcL segment (M1D and M1L) are non-specific amplification products. A 2 kb DNA ladder was used for fragment sizing. Arrows indicate the position of the 1400, 500 and 200 bp bands in the marker line. To establish whether there is a quantitative difference between the efficiency of the amplification of fragments, apparent bands were excised from the gel and the DNA purified and quantified from the excised fragments. Yields of the 1300 bp rbcL amplicon fragments were of 325 and 227.6 ng, 500 bp STS_8561_ palustris of 375 and 388 ng and 209 bp STS_8461_ palustris of 420 and 426 ng, for dark- and light-adapted samples, respectively. No bands were observed for any of the samples using as template the DNA isolated by method M2, suggesting no amplification of the target gene fragments. Abbreviations stand for: D = dark-adapted; L = light-adapted; M1 = method 1; M2 = method 2.
    Ethidium Bromide Stained 2, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 69 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Purification and in vitro cleavage assay of Synechocystis WT Cas6-1 and mutated Cas6-1 proteins. (A) Coomassie-stained 15% SDS-PAGE of heterologously overexpressed and purified Cas6-1 protein variants. The expected molecular weight of Cas6-1_6xHis is ∼ 30 kDa. 66.67 pmol recombinant enzyme was loaded on each lane and PageRuler™ Prestained Protein Ladder (Thermo Fisher Scientific) served for protein size estimation. (B, C) In in vitro cleavage assays both the (B) CRISPR1 RNA oligo repeat sequence and the (C) pre-acrRNA cassette is processed with variable efficiency by the purified Cas6-1 protein variants shown in (A). After incubation at 30°C the RNA fragments were electrophoretically size separated on a 15% PAA sequencing gel and visualized with SYBR® Gold Nucleic Acid Gel Stain. The processing results in a pattern of RNA fragments of distinct lengths. Cleavage assays were conducted with a three-fold excess (B) or a 1:1 ratio (C) of protein to RNA-substrate in cleavage buffer and incubated at 30°C for the indicated time. Expected RNA fragments and their corresponding sizes are illustrated by cartoons. Another Cas6-1 WT protein sample, which was overexpressed and purified independently, is indicated by an asterisk. Negative controls were complemented with 1 μL of H 2 O or 1 μL eluate of a mock control. Two additional nucleotides resulting from in vitro transcription were added to the 215 nt long acrRNA yielding a 217 nt long pre-acrRNA. For RNA size estimation a CRISPR1 repeat oligo ladder (C1L), RiboRuler low range RNA ladder (Thermo Fisher Scientific), low range ssRNA ladder or microRNA marker (both from New England Biolabs) were used.

    Journal: RNA Biology

    Article Title: Biochemical analysis of the Cas6-1 RNA endonuclease associated with the subtype I-D CRISPR-Cas system in Synechocystis sp. PCC 6803

    doi: 10.1080/15476286.2018.1447742

    Figure Lengend Snippet: Purification and in vitro cleavage assay of Synechocystis WT Cas6-1 and mutated Cas6-1 proteins. (A) Coomassie-stained 15% SDS-PAGE of heterologously overexpressed and purified Cas6-1 protein variants. The expected molecular weight of Cas6-1_6xHis is ∼ 30 kDa. 66.67 pmol recombinant enzyme was loaded on each lane and PageRuler™ Prestained Protein Ladder (Thermo Fisher Scientific) served for protein size estimation. (B, C) In in vitro cleavage assays both the (B) CRISPR1 RNA oligo repeat sequence and the (C) pre-acrRNA cassette is processed with variable efficiency by the purified Cas6-1 protein variants shown in (A). After incubation at 30°C the RNA fragments were electrophoretically size separated on a 15% PAA sequencing gel and visualized with SYBR® Gold Nucleic Acid Gel Stain. The processing results in a pattern of RNA fragments of distinct lengths. Cleavage assays were conducted with a three-fold excess (B) or a 1:1 ratio (C) of protein to RNA-substrate in cleavage buffer and incubated at 30°C for the indicated time. Expected RNA fragments and their corresponding sizes are illustrated by cartoons. Another Cas6-1 WT protein sample, which was overexpressed and purified independently, is indicated by an asterisk. Negative controls were complemented with 1 μL of H 2 O or 1 μL eluate of a mock control. Two additional nucleotides resulting from in vitro transcription were added to the 215 nt long acrRNA yielding a 217 nt long pre-acrRNA. For RNA size estimation a CRISPR1 repeat oligo ladder (C1L), RiboRuler low range RNA ladder (Thermo Fisher Scientific), low range ssRNA ladder or microRNA marker (both from New England Biolabs) were used.

    Article Snippet: The low range ssRNA ladder (ssRNA marker) (New England Biolabs), the RiboRuler low range RNA ladder (Thermo Fisher Scientific) and the microRNA marker (New England Biolabs) served as RNA ladders for size estimation.

    Techniques: Purification, In Vitro, Cleavage Assay, Staining, SDS Page, Molecular Weight, Recombinant, Sequencing, Incubation, Marker

    Incubation of in vitro transcripts of CRISPR1, 2 and 3 with Cas6-1. ( A ) A total of 2.3 μM of CRISPR1 transcript is cleaved by 6.2 μM Cas6-1 by incubation for 1 h. ( B ) A total of 2 μM of CRISPR2 transcript is cleaved by 6.2 μM Cas6-1 by incubation for 1 h. ( C ) A total of 3.2 μM of CRISPR3 transcript is not cleaved by 6.5 μM Cas6-1 by incubation for 2 h. ( D ) The cleavage of 6.6 μM of the shorter CRISPR1* in vitro transcript by 5.8 μM Cas6-1 monitored over a time of 2 h 20 min. Since no finally mature crRNA ( 17 ) was detected, the in vivo presence of an unknown ribonuclease is suggested. All reactions were performed at 37°C in a reaction volume of 5 μl. RNA was separated by 8 M urea 10% PAGE and bands were visualized by ethidium bromide staining. Diamonds represent repeats and rectangles spacer sequences. Transcripts were not gel purified, explaining the appearance of fragments smaller than the full length transcripts of CRISPR1 and 2 in absence of Cas6-1. Eb1, elution buffer 1 used as negative control. Molecular markers: MN, Low Range ssRNA Ladder (NEB); MF, RiboRuler Low Range RNA Ladder (Thermo Fisher Scientific).

    Journal: Nucleic Acids Research

    Article Title: Structural constraints and enzymatic promiscuity in the Cas6-dependent generation of crRNAs

    doi: 10.1093/nar/gkw786

    Figure Lengend Snippet: Incubation of in vitro transcripts of CRISPR1, 2 and 3 with Cas6-1. ( A ) A total of 2.3 μM of CRISPR1 transcript is cleaved by 6.2 μM Cas6-1 by incubation for 1 h. ( B ) A total of 2 μM of CRISPR2 transcript is cleaved by 6.2 μM Cas6-1 by incubation for 1 h. ( C ) A total of 3.2 μM of CRISPR3 transcript is not cleaved by 6.5 μM Cas6-1 by incubation for 2 h. ( D ) The cleavage of 6.6 μM of the shorter CRISPR1* in vitro transcript by 5.8 μM Cas6-1 monitored over a time of 2 h 20 min. Since no finally mature crRNA ( 17 ) was detected, the in vivo presence of an unknown ribonuclease is suggested. All reactions were performed at 37°C in a reaction volume of 5 μl. RNA was separated by 8 M urea 10% PAGE and bands were visualized by ethidium bromide staining. Diamonds represent repeats and rectangles spacer sequences. Transcripts were not gel purified, explaining the appearance of fragments smaller than the full length transcripts of CRISPR1 and 2 in absence of Cas6-1. Eb1, elution buffer 1 used as negative control. Molecular markers: MN, Low Range ssRNA Ladder (NEB); MF, RiboRuler Low Range RNA Ladder (Thermo Fisher Scientific).

    Article Snippet: Before loading onto denaturing 8 M urea 10% PAA gels, reactions were incubated at 95°C for 5 min. As size markers served the Low Range ssRNA Ladder from NEB (MN ), the RiboRuler Low Range RNA Ladder from Thermo Fisher Scientific (MF ) and the Low Molecular Weight Marker from Affymetrix (MA ).

    Techniques: Incubation, In Vitro, In Vivo, Polyacrylamide Gel Electrophoresis, Staining, Purification, Negative Control

    Electrophoretic analysis of partial pep A gene (596 bp) of B. pseudomallei digested with Stu I and Hinc II restriction analysis. (12% polyacrylamide gel, 1X TBE buffer, 8 V/cm, 130 min); Lane M- O’GeneRuler™ ultra low range DNA ladder; Lane 1- B. pseudomallei NCTC 13178; Lane 2- B. pseudomallei ATCC 23343; Lane 3- Type I; Lane 4- Type II; Lane 5- Type III.

    Journal: BMC Microbiology

    Article Title: Enzymatic and molecular characterisation of leucine aminopeptidase of Burkholderia pseudomallei

    doi: 10.1186/1471-2180-13-110

    Figure Lengend Snippet: Electrophoretic analysis of partial pep A gene (596 bp) of B. pseudomallei digested with Stu I and Hinc II restriction analysis. (12% polyacrylamide gel, 1X TBE buffer, 8 V/cm, 130 min); Lane M- O’GeneRuler™ ultra low range DNA ladder; Lane 1- B. pseudomallei NCTC 13178; Lane 2- B. pseudomallei ATCC 23343; Lane 3- Type I; Lane 4- Type II; Lane 5- Type III.

    Article Snippet: An O’GeneRuler™ Ultra Low Range DNA ladder (Fermentas, Lithuania) was used as molecular weight marker.

    Techniques:

    In vivo expression and SL trans -splicing of a chimeric TnI/β-gal mRNA from a TnI/β-gal gene construct lacking the SL sequence. ( A ) Expression of β-gal, revealed by X-Gal staining (blue), in tail muscle of embryos 12 h following introduction of CiTnILacZ(−1.5) DNA into zygotes by electroporation. ( B ) RT–PCR amplification of β-gal mRNA with β-gal-specific leftward priming, and rightward priming with the SL primer. (Lane 1 ) Size markers (MBI Fermentas ladder mix, 10–0.1 kb; top visible band, 3 kb, bottom visible band 500 bp). (Lanes 2,3 ) RT–PCR products from two different batches of transfected embryos. The template in lane 4 was tRNA, used as a carrier in embryo RNA isolations. A 550-bp product, the size predicted for SL-ended β-gal mRNA, was produced from both embryo batches. [Production of this product required the presence of both primers and did not occur when CiTnILacZ(−1.5) plasmid DNA was used as the amplification template. An additional product of ∼400 bp seen in lanes 2 and 3 required only the β-gal-specific primer and was produced in control amplifications of CiTnILacZ(−1.5) plasmid DNA; it apparently results from rightward mis-priming by the β-gal-specific primer upstream of its normal leftward priming site.] ( C ) DNA sequence of 550-bp RT–PCR product. The 550-bp product (as in B ) was recovered and sequenced using the SL primer ( right ) and β-gal-specific primer ( left ). The leftward sequence confirmed the presence of the SL primer (bold) immediately upstream of TnI mRNA nucleotide 17. Not shown is the Bam HI site engineered at the 5′-end of the SL primer. Sequences deriving from the β-gal reporter gene are shown in lower case.

    Journal: Genes & Development

    Article Title: mRNA 5?-leader trans-splicing in the chordates

    doi: 10.1101/gad.865401

    Figure Lengend Snippet: In vivo expression and SL trans -splicing of a chimeric TnI/β-gal mRNA from a TnI/β-gal gene construct lacking the SL sequence. ( A ) Expression of β-gal, revealed by X-Gal staining (blue), in tail muscle of embryos 12 h following introduction of CiTnILacZ(−1.5) DNA into zygotes by electroporation. ( B ) RT–PCR amplification of β-gal mRNA with β-gal-specific leftward priming, and rightward priming with the SL primer. (Lane 1 ) Size markers (MBI Fermentas ladder mix, 10–0.1 kb; top visible band, 3 kb, bottom visible band 500 bp). (Lanes 2,3 ) RT–PCR products from two different batches of transfected embryos. The template in lane 4 was tRNA, used as a carrier in embryo RNA isolations. A 550-bp product, the size predicted for SL-ended β-gal mRNA, was produced from both embryo batches. [Production of this product required the presence of both primers and did not occur when CiTnILacZ(−1.5) plasmid DNA was used as the amplification template. An additional product of ∼400 bp seen in lanes 2 and 3 required only the β-gal-specific primer and was produced in control amplifications of CiTnILacZ(−1.5) plasmid DNA; it apparently results from rightward mis-priming by the β-gal-specific primer upstream of its normal leftward priming site.] ( C ) DNA sequence of 550-bp RT–PCR product. The 550-bp product (as in B ) was recovered and sequenced using the SL primer ( right ) and β-gal-specific primer ( left ). The leftward sequence confirmed the presence of the SL primer (bold) immediately upstream of TnI mRNA nucleotide 17. Not shown is the Bam HI site engineered at the 5′-end of the SL primer. Sequences deriving from the β-gal reporter gene are shown in lower case.

    Article Snippet: RNA markers were the MBI Fermentas low-molecular weight RNA marker set.

    Techniques: In Vivo, Expressing, Construct, Sequencing, Staining, Electroporation, Reverse Transcription Polymerase Chain Reaction, Amplification, Transfection, Produced, Plasmid Preparation

    Genomic organization and expression strategy of Or-CD15 . ( A ) Screen shot of the RpL23A / Uhg7 region taken from the UCSC genome browser. The RpL23A-RA and Uhg7 transcripts are depicted in blue, the location of Or-CD15 and the newly identified transcript RpL23A-RB are shown in black. Below, sequence conservation as measured by phastcons and the coverage of a multiple genome alignment is indicated; ( B ) Northern blot analysis of adult fruit fly RNA. The genomic position of the utilised probe is outlined in ( A ). The band at about 100 nt corresponds to snoRNA Or-CD15 ; sizes are indicated by an RNA molecular weight ladder (RiboRuler Low Range, Life Technologies); ( C ) PCR experiments using primers annealing to RpL23A exon 1 and Uhg7 exon 4; amplification of the genomic DNA (gDNA) produces the expected fragment of about 2 kb; the same primers used in RT-PCR experiments successfully amplified a fragment of 1.2 kb, representative of the new RpL23A-RB transcript. In this transcript, RpL23A and Uhg7 sequences are fused to each other. On the right, a DNA molecular weight ladder (100bp DNA ladder, New England BioLabs, Ipswich, MA, USA) is given.

    Journal: Non-Coding RNA

    Article Title: Unusual Novel SnoRNA-Like RNAs in Drosophila melanogaster

    doi: 10.3390/ncrna1020139

    Figure Lengend Snippet: Genomic organization and expression strategy of Or-CD15 . ( A ) Screen shot of the RpL23A / Uhg7 region taken from the UCSC genome browser. The RpL23A-RA and Uhg7 transcripts are depicted in blue, the location of Or-CD15 and the newly identified transcript RpL23A-RB are shown in black. Below, sequence conservation as measured by phastcons and the coverage of a multiple genome alignment is indicated; ( B ) Northern blot analysis of adult fruit fly RNA. The genomic position of the utilised probe is outlined in ( A ). The band at about 100 nt corresponds to snoRNA Or-CD15 ; sizes are indicated by an RNA molecular weight ladder (RiboRuler Low Range, Life Technologies); ( C ) PCR experiments using primers annealing to RpL23A exon 1 and Uhg7 exon 4; amplification of the genomic DNA (gDNA) produces the expected fragment of about 2 kb; the same primers used in RT-PCR experiments successfully amplified a fragment of 1.2 kb, representative of the new RpL23A-RB transcript. In this transcript, RpL23A and Uhg7 sequences are fused to each other. On the right, a DNA molecular weight ladder (100bp DNA ladder, New England BioLabs, Ipswich, MA, USA) is given.

    Article Snippet: The size of RNAs was determined by using high range and low range RNA molecular weight markers (Life Technologies) Quantitative real-time RT-PCR (qRT-PCR) experiments were performed in triplicate using iQ5 Multicolor Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) as previously described [ ].

    Techniques: Expressing, Sequencing, Northern Blot, Molecular Weight, Polymerase Chain Reaction, Amplification, Reverse Transcription Polymerase Chain Reaction

    Incubation of in vitro transcripts of CRISPR1, 2 and 3 with Cas6-1. ( A ) A total of 2.3 μM of CRISPR1 transcript is cleaved by 6.2 μM Cas6-1 by incubation for 1 h. ( B ) A total of 2 μM of CRISPR2 transcript is cleaved by 6.2 μM Cas6-1 by incubation for 1 h. ( C ) A total of 3.2 μM of CRISPR3 transcript is not cleaved by 6.5 μM Cas6-1 by incubation for 2 h. ( D ) The cleavage of 6.6 μM of the shorter CRISPR1* in vitro transcript by 5.8 μM Cas6-1 monitored over a time of 2 h 20 min. Since no finally mature crRNA ( 17 ) was detected, the in vivo presence of an unknown ribonuclease is suggested. All reactions were performed at 37°C in a reaction volume of 5 μl. RNA was separated by 8 M urea 10% PAGE and bands were visualized by ethidium bromide staining. Diamonds represent repeats and rectangles spacer sequences. Transcripts were not gel purified, explaining the appearance of fragments smaller than the full length transcripts of CRISPR1 and 2 in absence of Cas6-1. Eb1, elution buffer 1 used as negative control. Molecular markers: MN, Low Range ssRNA Ladder (NEB); MF, RiboRuler Low Range RNA Ladder (Thermo Fisher Scientific).

    Journal: Nucleic Acids Research

    Article Title: Structural constraints and enzymatic promiscuity in the Cas6-dependent generation of crRNAs

    doi: 10.1093/nar/gkw786

    Figure Lengend Snippet: Incubation of in vitro transcripts of CRISPR1, 2 and 3 with Cas6-1. ( A ) A total of 2.3 μM of CRISPR1 transcript is cleaved by 6.2 μM Cas6-1 by incubation for 1 h. ( B ) A total of 2 μM of CRISPR2 transcript is cleaved by 6.2 μM Cas6-1 by incubation for 1 h. ( C ) A total of 3.2 μM of CRISPR3 transcript is not cleaved by 6.5 μM Cas6-1 by incubation for 2 h. ( D ) The cleavage of 6.6 μM of the shorter CRISPR1* in vitro transcript by 5.8 μM Cas6-1 monitored over a time of 2 h 20 min. Since no finally mature crRNA ( 17 ) was detected, the in vivo presence of an unknown ribonuclease is suggested. All reactions were performed at 37°C in a reaction volume of 5 μl. RNA was separated by 8 M urea 10% PAGE and bands were visualized by ethidium bromide staining. Diamonds represent repeats and rectangles spacer sequences. Transcripts were not gel purified, explaining the appearance of fragments smaller than the full length transcripts of CRISPR1 and 2 in absence of Cas6-1. Eb1, elution buffer 1 used as negative control. Molecular markers: MN, Low Range ssRNA Ladder (NEB); MF, RiboRuler Low Range RNA Ladder (Thermo Fisher Scientific).

    Article Snippet: Before loading onto denaturing 8 M urea 10% PAA gels, reactions were incubated at 95°C for 5 min. As size markers served the Low Range ssRNA Ladder from NEB (MN ), the RiboRuler Low Range RNA Ladder from Thermo Fisher Scientific (MF ) and the Low Molecular Weight Marker from Affymetrix (MA ).

    Techniques: Incubation, In Vitro, In Vivo, Polyacrylamide Gel Electrophoresis, Staining, Purification, Negative Control

    Amplification of selected target gene fragments from DNA extracted from in situ dark- or light-adapted mature pin oak ( Q. palustris ) leaves. The figure shows a representative example of PCR amplification of the 1300 bp rbcL , 500 bp STS_8561_ palustris and 209 bp STS_8461_ palustris sequence tagged site segments from dark- (D) and light-adapted (L) leaves, using extraction method M1 and M2, on a 2% agarose gel containing 0.5 µg/mL ethidium bromide. The low molecular weight bands (at about 175 bp) in the lane showing the amplified rbcL segment (M1D and M1L) are non-specific amplification products. A 2 kb DNA ladder was used for fragment sizing. Arrows indicate the position of the 1400, 500 and 200 bp bands in the marker line. To establish whether there is a quantitative difference between the efficiency of the amplification of fragments, apparent bands were excised from the gel and the DNA purified and quantified from the excised fragments. Yields of the 1300 bp rbcL amplicon fragments were of 325 and 227.6 ng, 500 bp STS_8561_ palustris of 375 and 388 ng and 209 bp STS_8461_ palustris of 420 and 426 ng, for dark- and light-adapted samples, respectively. No bands were observed for any of the samples using as template the DNA isolated by method M2, suggesting no amplification of the target gene fragments. Abbreviations stand for: D = dark-adapted; L = light-adapted; M1 = method 1; M2 = method 2.

    Journal: Plants

    Article Title: In Situ Dark Adaptation Enhances the Efficiency of DNA Extraction from Mature Pin Oak (Quercus palustris) Leaves, Facilitating the Identification of Partial Sequences of the 18S rRNA and Isoprene Synthase (IspS) Genes

    doi: 10.3390/plants6040052

    Figure Lengend Snippet: Amplification of selected target gene fragments from DNA extracted from in situ dark- or light-adapted mature pin oak ( Q. palustris ) leaves. The figure shows a representative example of PCR amplification of the 1300 bp rbcL , 500 bp STS_8561_ palustris and 209 bp STS_8461_ palustris sequence tagged site segments from dark- (D) and light-adapted (L) leaves, using extraction method M1 and M2, on a 2% agarose gel containing 0.5 µg/mL ethidium bromide. The low molecular weight bands (at about 175 bp) in the lane showing the amplified rbcL segment (M1D and M1L) are non-specific amplification products. A 2 kb DNA ladder was used for fragment sizing. Arrows indicate the position of the 1400, 500 and 200 bp bands in the marker line. To establish whether there is a quantitative difference between the efficiency of the amplification of fragments, apparent bands were excised from the gel and the DNA purified and quantified from the excised fragments. Yields of the 1300 bp rbcL amplicon fragments were of 325 and 227.6 ng, 500 bp STS_8561_ palustris of 375 and 388 ng and 209 bp STS_8461_ palustris of 420 and 426 ng, for dark- and light-adapted samples, respectively. No bands were observed for any of the samples using as template the DNA isolated by method M2, suggesting no amplification of the target gene fragments. Abbreviations stand for: D = dark-adapted; L = light-adapted; M1 = method 1; M2 = method 2.

    Article Snippet: The amplified PCR products were separated on a 2% agarose gel and stained with 0.5 µg/mL ethidium bromide (Sigma-Aldrich, St. Louis, MO, USA) and the bands were imaged with a CareStream Gel Logic 2200 Pro Imaging system (CareStream Health Solutions, Rochester, NY, USA).

    Techniques: Amplification, In Situ, Polymerase Chain Reaction, Sequencing, Agarose Gel Electrophoresis, Molecular Weight, Marker, Purification, Isolation

    Amplification of a partial segment of the putative gene encoding for the isoprene synthase (IspS) enzyme. PCR products were separated on a 2% agarose gel stained with 0.5 µg/mL ethidium bromide. The arrow indicates the position of the 165 bp fragment of the putative IspS gene. Lanes 2 and 3 were loaded with representative reactions containing DNA templates isolated from dark-adapted leaves, using M1. Lanes 4 and 5 were loaded with representative reactions containing DNA templates isolated from light-adapted leaves, using M1. The white arrow annotates the position of the 165 bp fragments. D = dark-adapted; L = light-adapted.

    Journal: Plants

    Article Title: In Situ Dark Adaptation Enhances the Efficiency of DNA Extraction from Mature Pin Oak (Quercus palustris) Leaves, Facilitating the Identification of Partial Sequences of the 18S rRNA and Isoprene Synthase (IspS) Genes

    doi: 10.3390/plants6040052

    Figure Lengend Snippet: Amplification of a partial segment of the putative gene encoding for the isoprene synthase (IspS) enzyme. PCR products were separated on a 2% agarose gel stained with 0.5 µg/mL ethidium bromide. The arrow indicates the position of the 165 bp fragment of the putative IspS gene. Lanes 2 and 3 were loaded with representative reactions containing DNA templates isolated from dark-adapted leaves, using M1. Lanes 4 and 5 were loaded with representative reactions containing DNA templates isolated from light-adapted leaves, using M1. The white arrow annotates the position of the 165 bp fragments. D = dark-adapted; L = light-adapted.

    Article Snippet: The amplified PCR products were separated on a 2% agarose gel and stained with 0.5 µg/mL ethidium bromide (Sigma-Aldrich, St. Louis, MO, USA) and the bands were imaged with a CareStream Gel Logic 2200 Pro Imaging system (CareStream Health Solutions, Rochester, NY, USA).

    Techniques: Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, Isolation