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  • 96
    Thermo Fisher rapid dna ligation
    SCIPay design permits CRISPR-guided integration of specific donor <t>DNA</t> sequences via homology directed repair. (A) Overview of SCIPay design. Here, HA present in the payload sequence lead to its genomic integration via HDR. (B – G) EL4 cells were electroporated with SCIPay containing a <t>BFP</t> transgene targeting B2M with various HA lengths. (B) Percentage of knock-out cells. (C) Efficiency and (D) precision scores for GFP, denoting integration of the non-payload plasmid sequence. (E) Efficiency and (F) precision scores for BFP, denoting HDR integration of BFP. (G) Ratio of BFP-to GFP-positive cells. (H) Representative flow cytometry plots. Results in (B – G) represent means +/- SEM of 4 independent experiments. Data was analyzed using 1-way ANOVA and significant differences are indicated with lower case letters, where groups with different letters are significantly different than each other (p
    Rapid Dna Ligation, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 516 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    addgene inc rapid dna ligation kit
    SCIPay design permits CRISPR-guided integration of specific donor <t>DNA</t> sequences via homology directed repair. (A) Overview of SCIPay design. Here, HA present in the payload sequence lead to its genomic integration via HDR. (B – G) EL4 cells were electroporated with SCIPay containing a <t>BFP</t> transgene targeting B2M with various HA lengths. (B) Percentage of knock-out cells. (C) Efficiency and (D) precision scores for GFP, denoting integration of the non-payload plasmid sequence. (E) Efficiency and (F) precision scores for BFP, denoting HDR integration of BFP. (G) Ratio of BFP-to GFP-positive cells. (H) Representative flow cytometry plots. Results in (B – G) represent means +/- SEM of 4 independent experiments. Data was analyzed using 1-way ANOVA and significant differences are indicated with lower case letters, where groups with different letters are significantly different than each other (p
    Rapid Dna Ligation Kit, supplied by addgene inc, 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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    Thermo Fisher t4 dna ligase
    SCIPay design permits CRISPR-guided integration of specific donor <t>DNA</t> sequences via homology directed repair. (A) Overview of SCIPay design. Here, HA present in the payload sequence lead to its genomic integration via HDR. (B – G) EL4 cells were electroporated with SCIPay containing a <t>BFP</t> transgene targeting B2M with various HA lengths. (B) Percentage of knock-out cells. (C) Efficiency and (D) precision scores for GFP, denoting integration of the non-payload plasmid sequence. (E) Efficiency and (F) precision scores for BFP, denoting HDR integration of BFP. (G) Ratio of BFP-to GFP-positive cells. (H) Representative flow cytometry plots. Results in (B – G) represent means +/- SEM of 4 independent experiments. Data was analyzed using 1-way ANOVA and significant differences are indicated with lower case letters, where groups with different letters are significantly different than each other (p
    T4 Dna Ligase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 25057 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher first strand cdna synthesis
    SCIPay design permits CRISPR-guided integration of specific donor <t>DNA</t> sequences via homology directed repair. (A) Overview of SCIPay design. Here, HA present in the payload sequence lead to its genomic integration via HDR. (B – G) EL4 cells were electroporated with SCIPay containing a <t>BFP</t> transgene targeting B2M with various HA lengths. (B) Percentage of knock-out cells. (C) Efficiency and (D) precision scores for GFP, denoting integration of the non-payload plasmid sequence. (E) Efficiency and (F) precision scores for BFP, denoting HDR integration of BFP. (G) Ratio of BFP-to GFP-positive cells. (H) Representative flow cytometry plots. Results in (B – G) represent means +/- SEM of 4 independent experiments. Data was analyzed using 1-way ANOVA and significant differences are indicated with lower case letters, where groups with different letters are significantly different than each other (p
    First Strand Cdna Synthesis, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 29583 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher cdna ends
    Schematic illustrating the preparation and analysis of circularized <t>RNA</t> libraries. Purified RNA was treated with a thermostable single-stranded nucleic acid-specific ligase (CircLigase II) to achieve intramolecular ligation of individual molecules. After ligation any remaining linear RNA was degraded using RNase R and the circular RNAs were reverse transcribed using random primers incorporating an adaptor sequence for subsequent sequencing. A second adaptor sequence was added by extension of the <t>cDNA</t> using a 3΄-end-blocked oligonucleotide as a template. The cDNA was then amplified by limited PCR. Primers were designed to incorporate sequences for subsequent deep sequencing.
    Cdna Ends, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 9717 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher cdna ends race pcr
    Generation and in vitro characterization of recombinant NIEV. (A) Schematic drawing of the NIEV genome organization and reverse genetics system. Depicted nucleotide positions indicate the borders of the encoded viral proteins or the genome end. Below the NIEV genome organization, the reverse-transcribed NIEV <t>cDNA</t> fragments, including the restriction sites used for cloning, are shown. An SP6 promoter sequence was fused to the 5′ end of the NIEV genome. EcoRI indicates the silent genetic marker mutation resulting in deletion of an EcoRI restriction site. After assembly of the cDNA fragments into two plasmids, in vitro ligation was performed followed by EagI linearization of the ligation product. The latter was transcribed in vitro , and the transcribed RNA was electroporated into C6/36 cells to recover rec NIEV. (B) Plaque morphology of wt NIEV and rec NIEV. Plaque morphology was analyzed by ICA in C6/36 cells using a tragacanth overlay. At 7 days postelectroporation, cells were fixed and subjected to crystal violet staining. (C) Growth kinetics of wt NIEV and rec NIEV. C6/36 cells were infected at an MOI of 0.1. Quantification of viral genome copies in the supernatant was performed by real-time <t>PCR.</t> Data represent means and ranges of results of duplicate infection experiments. (D) Verification of the genetic marker introduced into rec NIEV. Viral RNA was isolated from supernatants of cells infected with the indicated viruses and used as the template for RT-PCR spanning the region containing the deleted EcoRI site in rec NIEV. RT-PCR products were loaded either directly (−) or after EcoRI restriction (+) on an ethidium bromide-stained agarose gel.
    Cdna Ends Race Pcr, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 198 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    SCIPay design permits CRISPR-guided integration of specific donor DNA sequences via homology directed repair. (A) Overview of SCIPay design. Here, HA present in the payload sequence lead to its genomic integration via HDR. (B – G) EL4 cells were electroporated with SCIPay containing a BFP transgene targeting B2M with various HA lengths. (B) Percentage of knock-out cells. (C) Efficiency and (D) precision scores for GFP, denoting integration of the non-payload plasmid sequence. (E) Efficiency and (F) precision scores for BFP, denoting HDR integration of BFP. (G) Ratio of BFP-to GFP-positive cells. (H) Representative flow cytometry plots. Results in (B – G) represent means +/- SEM of 4 independent experiments. Data was analyzed using 1-way ANOVA and significant differences are indicated with lower case letters, where groups with different letters are significantly different than each other (p

    Journal: bioRxiv

    Article Title: Self-cutting and integrating CRISPR plasmids (SCIPs) enable targeted genomic integration of large genetic payloads for rapid cell engineering

    doi: 10.1101/2020.03.25.008276

    Figure Lengend Snippet: SCIPay design permits CRISPR-guided integration of specific donor DNA sequences via homology directed repair. (A) Overview of SCIPay design. Here, HA present in the payload sequence lead to its genomic integration via HDR. (B – G) EL4 cells were electroporated with SCIPay containing a BFP transgene targeting B2M with various HA lengths. (B) Percentage of knock-out cells. (C) Efficiency and (D) precision scores for GFP, denoting integration of the non-payload plasmid sequence. (E) Efficiency and (F) precision scores for BFP, denoting HDR integration of BFP. (G) Ratio of BFP-to GFP-positive cells. (H) Representative flow cytometry plots. Results in (B – G) represent means +/- SEM of 4 independent experiments. Data was analyzed using 1-way ANOVA and significant differences are indicated with lower case letters, where groups with different letters are significantly different than each other (p

    Article Snippet: This modular plasmid allows insertion of gRNA-coding DNA oligonucleotides using BpiI as outlined above and subsequent cloning of HA- and bait-coding DNA fragments engineered to be in-frame with the preceding genome sequence and following P2A-BFP sequence with BsaI using Gibson or restriction/ligation cloning.

    Techniques: CRISPR, Sequencing, Knock-Out, Plasmid Preparation, Flow Cytometry

    Schematic illustrating the preparation and analysis of circularized RNA libraries. Purified RNA was treated with a thermostable single-stranded nucleic acid-specific ligase (CircLigase II) to achieve intramolecular ligation of individual molecules. After ligation any remaining linear RNA was degraded using RNase R and the circular RNAs were reverse transcribed using random primers incorporating an adaptor sequence for subsequent sequencing. A second adaptor sequence was added by extension of the cDNA using a 3΄-end-blocked oligonucleotide as a template. The cDNA was then amplified by limited PCR. Primers were designed to incorporate sequences for subsequent deep sequencing.

    Journal: Nucleic Acids Research

    Article Title: Simultaneous processing and degradation of mitochondrial RNAs revealed by circularized RNA sequencing

    doi: 10.1093/nar/gkx104

    Figure Lengend Snippet: Schematic illustrating the preparation and analysis of circularized RNA libraries. Purified RNA was treated with a thermostable single-stranded nucleic acid-specific ligase (CircLigase II) to achieve intramolecular ligation of individual molecules. After ligation any remaining linear RNA was degraded using RNase R and the circular RNAs were reverse transcribed using random primers incorporating an adaptor sequence for subsequent sequencing. A second adaptor sequence was added by extension of the cDNA using a 3΄-end-blocked oligonucleotide as a template. The cDNA was then amplified by limited PCR. Primers were designed to incorporate sequences for subsequent deep sequencing.

    Article Snippet: Rapid amplification of cDNA ends (RACE) The 5΄ ends of RNA extracted from wild-type and Mrpp3 knockout hearts were captured using the RLM-RACE kit (Ambion, Thermo Fisher Scientific), according to the manufacturer's recommendations, except the calf intestine alkaline phosphatase and tobacco acid pyrophosphatase treatments were omitted since mitochondrial RNAs are not capped.

    Techniques: Purification, Ligation, Sequencing, Amplification, Polymerase Chain Reaction

    Polyadenylation of mRNA decay intermediates. ( A ) 5΄ rapid amplification of cDNA ends (5΄-RACE) validates the 5΄ ends of mt-Co1 . 3΄-RACE identified total ( B ) and polyadenylated ( C ) processing intermediates that include mt-Co1 . Circularized RNA sequencing captured authentic small RNAs ( D ). The abundance of small RNAs (sRNAs) of different sizes identified by circularized RNA sequencing were compared to those detected by classical sRNA sequencing. The positions of the 19 and 22 nt sRNAs previously identified in the human mitochondrial transcriptome ( 27 ) are indicated by arrows. ( E ) Locations of sRNA 5΄ and 3΄ ends within mt-Co1 and the fold change between wild-type and Mrpp3 knockout mitochondrial circularized RNA libraries are shown. Small RNAs derived from mt-Co1 were increased in abundance in the Mrpp3 RNA libraries (Student's t -test, P = 0.0034). ( F ) A proportion of sRNA degradation products derived from mRNAs are polyadenylated. The proportion of polyadenylated sRNAs derived from the following mRNAs were significantly increased upon loss of MRPP3 (Student's t -test, P ≤ 0.05, indicated by an asterisk): mt-Co1, mt-Co2, mt-Atp8/6, mt-Co3, mt-Nd6 and mt-Cytb .

    Journal: Nucleic Acids Research

    Article Title: Simultaneous processing and degradation of mitochondrial RNAs revealed by circularized RNA sequencing

    doi: 10.1093/nar/gkx104

    Figure Lengend Snippet: Polyadenylation of mRNA decay intermediates. ( A ) 5΄ rapid amplification of cDNA ends (5΄-RACE) validates the 5΄ ends of mt-Co1 . 3΄-RACE identified total ( B ) and polyadenylated ( C ) processing intermediates that include mt-Co1 . Circularized RNA sequencing captured authentic small RNAs ( D ). The abundance of small RNAs (sRNAs) of different sizes identified by circularized RNA sequencing were compared to those detected by classical sRNA sequencing. The positions of the 19 and 22 nt sRNAs previously identified in the human mitochondrial transcriptome ( 27 ) are indicated by arrows. ( E ) Locations of sRNA 5΄ and 3΄ ends within mt-Co1 and the fold change between wild-type and Mrpp3 knockout mitochondrial circularized RNA libraries are shown. Small RNAs derived from mt-Co1 were increased in abundance in the Mrpp3 RNA libraries (Student's t -test, P = 0.0034). ( F ) A proportion of sRNA degradation products derived from mRNAs are polyadenylated. The proportion of polyadenylated sRNAs derived from the following mRNAs were significantly increased upon loss of MRPP3 (Student's t -test, P ≤ 0.05, indicated by an asterisk): mt-Co1, mt-Co2, mt-Atp8/6, mt-Co3, mt-Nd6 and mt-Cytb .

    Article Snippet: Rapid amplification of cDNA ends (RACE) The 5΄ ends of RNA extracted from wild-type and Mrpp3 knockout hearts were captured using the RLM-RACE kit (Ambion, Thermo Fisher Scientific), according to the manufacturer's recommendations, except the calf intestine alkaline phosphatase and tobacco acid pyrophosphatase treatments were omitted since mitochondrial RNAs are not capped.

    Techniques: Rapid Amplification of cDNA Ends, RNA Sequencing Assay, Sequencing, Knock-Out, Derivative Assay

    Generation and in vitro characterization of recombinant NIEV. (A) Schematic drawing of the NIEV genome organization and reverse genetics system. Depicted nucleotide positions indicate the borders of the encoded viral proteins or the genome end. Below the NIEV genome organization, the reverse-transcribed NIEV cDNA fragments, including the restriction sites used for cloning, are shown. An SP6 promoter sequence was fused to the 5′ end of the NIEV genome. EcoRI indicates the silent genetic marker mutation resulting in deletion of an EcoRI restriction site. After assembly of the cDNA fragments into two plasmids, in vitro ligation was performed followed by EagI linearization of the ligation product. The latter was transcribed in vitro , and the transcribed RNA was electroporated into C6/36 cells to recover rec NIEV. (B) Plaque morphology of wt NIEV and rec NIEV. Plaque morphology was analyzed by ICA in C6/36 cells using a tragacanth overlay. At 7 days postelectroporation, cells were fixed and subjected to crystal violet staining. (C) Growth kinetics of wt NIEV and rec NIEV. C6/36 cells were infected at an MOI of 0.1. Quantification of viral genome copies in the supernatant was performed by real-time PCR. Data represent means and ranges of results of duplicate infection experiments. (D) Verification of the genetic marker introduced into rec NIEV. Viral RNA was isolated from supernatants of cells infected with the indicated viruses and used as the template for RT-PCR spanning the region containing the deleted EcoRI site in rec NIEV. RT-PCR products were loaded either directly (−) or after EcoRI restriction (+) on an ethidium bromide-stained agarose gel.

    Journal: mSphere

    Article Title: Host Range Restriction of Insect-Specific Flaviviruses Occurs at Several Levels of the Viral Life Cycle

    doi: 10.1128/mSphere.00375-16

    Figure Lengend Snippet: Generation and in vitro characterization of recombinant NIEV. (A) Schematic drawing of the NIEV genome organization and reverse genetics system. Depicted nucleotide positions indicate the borders of the encoded viral proteins or the genome end. Below the NIEV genome organization, the reverse-transcribed NIEV cDNA fragments, including the restriction sites used for cloning, are shown. An SP6 promoter sequence was fused to the 5′ end of the NIEV genome. EcoRI indicates the silent genetic marker mutation resulting in deletion of an EcoRI restriction site. After assembly of the cDNA fragments into two plasmids, in vitro ligation was performed followed by EagI linearization of the ligation product. The latter was transcribed in vitro , and the transcribed RNA was electroporated into C6/36 cells to recover rec NIEV. (B) Plaque morphology of wt NIEV and rec NIEV. Plaque morphology was analyzed by ICA in C6/36 cells using a tragacanth overlay. At 7 days postelectroporation, cells were fixed and subjected to crystal violet staining. (C) Growth kinetics of wt NIEV and rec NIEV. C6/36 cells were infected at an MOI of 0.1. Quantification of viral genome copies in the supernatant was performed by real-time PCR. Data represent means and ranges of results of duplicate infection experiments. (D) Verification of the genetic marker introduced into rec NIEV. Viral RNA was isolated from supernatants of cells infected with the indicated viruses and used as the template for RT-PCR spanning the region containing the deleted EcoRI site in rec NIEV. RT-PCR products were loaded either directly (−) or after EcoRI restriction (+) on an ethidium bromide-stained agarose gel.

    Article Snippet: Genome termini were sequenced by rapid amplification of cDNA ends (RACE)-PCR according to the instructions of the manufacturer (Invitrogen, Germany).

    Techniques: In Vitro, Recombinant, Clone Assay, Sequencing, Marker, Mutagenesis, Ligation, Staining, Infection, Real-time Polymerase Chain Reaction, Isolation, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis