turbo dna free kit  (Thermo Fisher)


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    Name:
    TURBO DNA free Kit
    Description:
    The TURBO DNA free Kit contains reagents for the efficient complete digestion of DNA along with the removal of the enzyme and divalent cations post digestion Note if you would like to purchase the enzyme alone without the inactivation and cation removal reagents please see TURBO DNAase Features of the TURBO DNA free Kit include • Hyperactive TURBO DNase is a catalytically superior enzyme compared to wild type DNase I• Removes trace quantities of DNA that can interfere with RT PCR• Reagent included to completely remove DNase without phenol treatment or heatingThe best method for genomic DNA removal prior to RT PCRTURBO DNase is a recombinant engineered form of DNase I that is much more efficient than wild type DNase I in digesting away trace amounts of unwanted DNA TURBO DNase binds DNA substrates 6 fold more tightly than traditional DNase I making this enzyme the tool of choice for clearing residual DNA that can generate a false positive signal in RT PCR applications TURBO DNase now includes an enhancer that increases the effectiveness by two orders of magnitude Efficient DNase and divalent cation removal without organic extraction or precipitationConventional DNase treatment of RNA samples prior to RT PCR typically call for inactivation of the DNase by phenol CHCl3 extraction or heating followed by a precipitation step to concentrate the RNA Phenol CHCl3 extractions can be cumbersome and time consuming Heating the sample to inactivate DNase can lead to chemical degradation of the RNA by divalent cations present in the DNase buffer The TURBO DNA free Kit circumvents these problems using a novel DNase Inactivation Reagent In addition to removing the TURBO DNase from the reaction the Inactivation Reagent also binds and removes divalent cations from the TURBO DNase Reaction Buffer
    Catalog Number:
    am1907
    Price:
    None
    Category:
    Proteins Enzymes Peptides
    Applications:
    General Real-Time PCR Reagents|PCR & Real-Time PCR|Real Time PCR (qPCR)|Reverse Transcription
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    Structured Review

    Thermo Fisher turbo dna free kit
    Overview of EV-BEAMing. <t>EVs</t> from serum or CSF samples were pelleted at 100,000 g for 80 minutes and processed as follows: (1) RNA was extracted and (2) analyzed for total yield and quality using the Bioanalyzer (Agilent). (3) RNA was then reverse transcribed into cDNA and 1/2 of the sample was used to determine the IDH1 cDNA copy number inside EVs by (4) qPCR analysis. (5) The remaining sample was preamplified (14 cycles) and used as input for BEAMing PCR. The resulting <t>DNA-coated</t> beads were interrogated with sequence-specific fluorescent probes to produce beads with wild-type (green) and mutant (red) profiles. (6) The percentage of beads with mutant DNA was determined by FACS and used in conjunction with the qPCR data to determine the minimum number of copies present to allow reliable detection of the mutant message.
    The TURBO DNA free Kit contains reagents for the efficient complete digestion of DNA along with the removal of the enzyme and divalent cations post digestion Note if you would like to purchase the enzyme alone without the inactivation and cation removal reagents please see TURBO DNAase Features of the TURBO DNA free Kit include • Hyperactive TURBO DNase is a catalytically superior enzyme compared to wild type DNase I• Removes trace quantities of DNA that can interfere with RT PCR• Reagent included to completely remove DNase without phenol treatment or heatingThe best method for genomic DNA removal prior to RT PCRTURBO DNase is a recombinant engineered form of DNase I that is much more efficient than wild type DNase I in digesting away trace amounts of unwanted DNA TURBO DNase binds DNA substrates 6 fold more tightly than traditional DNase I making this enzyme the tool of choice for clearing residual DNA that can generate a false positive signal in RT PCR applications TURBO DNase now includes an enhancer that increases the effectiveness by two orders of magnitude Efficient DNase and divalent cation removal without organic extraction or precipitationConventional DNase treatment of RNA samples prior to RT PCR typically call for inactivation of the DNase by phenol CHCl3 extraction or heating followed by a precipitation step to concentrate the RNA Phenol CHCl3 extractions can be cumbersome and time consuming Heating the sample to inactivate DNase can lead to chemical degradation of the RNA by divalent cations present in the DNase buffer The TURBO DNA free Kit circumvents these problems using a novel DNase Inactivation Reagent In addition to removing the TURBO DNase from the reaction the Inactivation Reagent also binds and removes divalent cations from the TURBO DNase Reaction Buffer
    https://www.bioz.com/result/turbo dna free kit/product/Thermo Fisher
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    turbo dna free kit - by Bioz Stars, 2021-03
    86/100 stars

    Images

    1) Product Images from "BEAMing and Droplet Digital PCR Analysis of Mutant IDH1 mRNA in Glioma Patient Serum and Cerebrospinal Fluid Extracellular Vesicles"

    Article Title: BEAMing and Droplet Digital PCR Analysis of Mutant IDH1 mRNA in Glioma Patient Serum and Cerebrospinal Fluid Extracellular Vesicles

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1038/mtna.2013.28

    Overview of EV-BEAMing. EVs from serum or CSF samples were pelleted at 100,000 g for 80 minutes and processed as follows: (1) RNA was extracted and (2) analyzed for total yield and quality using the Bioanalyzer (Agilent). (3) RNA was then reverse transcribed into cDNA and 1/2 of the sample was used to determine the IDH1 cDNA copy number inside EVs by (4) qPCR analysis. (5) The remaining sample was preamplified (14 cycles) and used as input for BEAMing PCR. The resulting DNA-coated beads were interrogated with sequence-specific fluorescent probes to produce beads with wild-type (green) and mutant (red) profiles. (6) The percentage of beads with mutant DNA was determined by FACS and used in conjunction with the qPCR data to determine the minimum number of copies present to allow reliable detection of the mutant message.
    Figure Legend Snippet: Overview of EV-BEAMing. EVs from serum or CSF samples were pelleted at 100,000 g for 80 minutes and processed as follows: (1) RNA was extracted and (2) analyzed for total yield and quality using the Bioanalyzer (Agilent). (3) RNA was then reverse transcribed into cDNA and 1/2 of the sample was used to determine the IDH1 cDNA copy number inside EVs by (4) qPCR analysis. (5) The remaining sample was preamplified (14 cycles) and used as input for BEAMing PCR. The resulting DNA-coated beads were interrogated with sequence-specific fluorescent probes to produce beads with wild-type (green) and mutant (red) profiles. (6) The percentage of beads with mutant DNA was determined by FACS and used in conjunction with the qPCR data to determine the minimum number of copies present to allow reliable detection of the mutant message.

    Techniques Used: Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Sequencing, Mutagenesis, FACS

    Related Articles

    Synthesized:

    Article Title: Regulatory mechanisms for 3?-end alternative splicing and polyadenylation of the Glial Fibrillary Acidic Protein, GFAP, transcript
    Article Snippet: Forty-eight hours after transfection the media was replaced with fresh medium, and 1 h after cells were transfected a second time, with either the GFAPmE7p(A) minigene alone or together with siRNA directed against Rrp6 or an irrelevant target as control, using Lipofectamine 2000 (Invitrogen). .. The cells were harvested after another 48 h. Five micrograms total RNA was treated with DNase (Turbo DNA-free kit, Ambion). cDNA was synthesized from 0.5 μg DNase treated RNA in 20 μ reactions using iScript™ cDNA synthesis Kit. ..

    Article Title: Efficient Editing of Malaria Parasite Genome Using the CRISPR/Cas9 System
    Article Snippet: RNA quantitation was done using Nanodrop (Thermo). .. Purified RNA was treated with DNase using Turbo DNA-free kit (Life Technologies). cDNA was synthesized using RevertAid reverse transcriptase (Fermentas). ..

    Isolation:

    Article Title: HEATR2 Plays a Conserved Role in Assembly of the Ciliary Motile Apparatus
    Article Snippet: Signal was detected using ECL-Prime detection kit (GE Healthcare) and either exposed to photographic film (Kodak Biomax XAR Film) or used for digital quantification on the ImageQuant LAS 4000 (GE Healthcare) according to the manufacturer's instructions. .. Total RNA was isolated according to manufacturer's protocol using RNAeasy minicolumns (Qiagen), followed by DNase treatment with Turbo DNA-free kit (Ambion). cDNA was made using First Strand Synthesis of cDNA for RT-PCR (AMV) kit (Roche). .. Standard RT-PCR was performed using primers spanning coding HEATR2 transcripts ENST00000297440 and predicted ENST00000313147 which uses an alternate 3′ UTR as well as final 13th exon ( ).

    Article Title: Opossum carboxylesterases: sequences, phylogeny and evidence for CES gene duplication events predating the marsupial-eutherian common ancestor
    Article Snippet: Sequencing was performed using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA), and nucleotide sequences were analyzed on an ABI Prism 3100 DNA sequencer (Applied Biosystems). .. RT-PCR Expression Studies and Sequencing of RT-PCR Products Total RNA was isolated from livers and small intestines of opossums using the TRI Reagent (Molecular Research Center, Cincinnati, OH), and treated with DNase I from the Turbo DNA-free kit (Applied Biosystems) according to the manufacturer's protocol to remove contaminating DNA from the RNA preparations. .. DNase I-treated RNA was reverse transcribed into cDNA using a High Capacity cDNA Reverse Transcription kit (Applied Biosystems).

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: HEATR2 Plays a Conserved Role in Assembly of the Ciliary Motile Apparatus
    Article Snippet: Signal was detected using ECL-Prime detection kit (GE Healthcare) and either exposed to photographic film (Kodak Biomax XAR Film) or used for digital quantification on the ImageQuant LAS 4000 (GE Healthcare) according to the manufacturer's instructions. .. Total RNA was isolated according to manufacturer's protocol using RNAeasy minicolumns (Qiagen), followed by DNase treatment with Turbo DNA-free kit (Ambion). cDNA was made using First Strand Synthesis of cDNA for RT-PCR (AMV) kit (Roche). .. Standard RT-PCR was performed using primers spanning coding HEATR2 transcripts ENST00000297440 and predicted ENST00000313147 which uses an alternate 3′ UTR as well as final 13th exon ( ).

    Article Title: Opossum carboxylesterases: sequences, phylogeny and evidence for CES gene duplication events predating the marsupial-eutherian common ancestor
    Article Snippet: Sequencing was performed using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA), and nucleotide sequences were analyzed on an ABI Prism 3100 DNA sequencer (Applied Biosystems). .. RT-PCR Expression Studies and Sequencing of RT-PCR Products Total RNA was isolated from livers and small intestines of opossums using the TRI Reagent (Molecular Research Center, Cincinnati, OH), and treated with DNase I from the Turbo DNA-free kit (Applied Biosystems) according to the manufacturer's protocol to remove contaminating DNA from the RNA preparations. .. DNase I-treated RNA was reverse transcribed into cDNA using a High Capacity cDNA Reverse Transcription kit (Applied Biosystems).

    Expressing:

    Article Title: Opossum carboxylesterases: sequences, phylogeny and evidence for CES gene duplication events predating the marsupial-eutherian common ancestor
    Article Snippet: Sequencing was performed using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA), and nucleotide sequences were analyzed on an ABI Prism 3100 DNA sequencer (Applied Biosystems). .. RT-PCR Expression Studies and Sequencing of RT-PCR Products Total RNA was isolated from livers and small intestines of opossums using the TRI Reagent (Molecular Research Center, Cincinnati, OH), and treated with DNase I from the Turbo DNA-free kit (Applied Biosystems) according to the manufacturer's protocol to remove contaminating DNA from the RNA preparations. .. DNase I-treated RNA was reverse transcribed into cDNA using a High Capacity cDNA Reverse Transcription kit (Applied Biosystems).

    Sequencing:

    Article Title: Opossum carboxylesterases: sequences, phylogeny and evidence for CES gene duplication events predating the marsupial-eutherian common ancestor
    Article Snippet: Sequencing was performed using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA), and nucleotide sequences were analyzed on an ABI Prism 3100 DNA sequencer (Applied Biosystems). .. RT-PCR Expression Studies and Sequencing of RT-PCR Products Total RNA was isolated from livers and small intestines of opossums using the TRI Reagent (Molecular Research Center, Cincinnati, OH), and treated with DNase I from the Turbo DNA-free kit (Applied Biosystems) according to the manufacturer's protocol to remove contaminating DNA from the RNA preparations. .. DNase I-treated RNA was reverse transcribed into cDNA using a High Capacity cDNA Reverse Transcription kit (Applied Biosystems).

    Purification:

    Article Title: Efficient Editing of Malaria Parasite Genome Using the CRISPR/Cas9 System
    Article Snippet: RNA quantitation was done using Nanodrop (Thermo). .. Purified RNA was treated with DNase using Turbo DNA-free kit (Life Technologies). cDNA was synthesized using RevertAid reverse transcriptase (Fermentas). ..

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    Thermo Fisher turbo dna free kit
    Overview of EV-BEAMing. <t>EVs</t> from serum or CSF samples were pelleted at 100,000 g for 80 minutes and processed as follows: (1) RNA was extracted and (2) analyzed for total yield and quality using the Bioanalyzer (Agilent). (3) RNA was then reverse transcribed into cDNA and 1/2 of the sample was used to determine the IDH1 cDNA copy number inside EVs by (4) qPCR analysis. (5) The remaining sample was preamplified (14 cycles) and used as input for BEAMing PCR. The resulting <t>DNA-coated</t> beads were interrogated with sequence-specific fluorescent probes to produce beads with wild-type (green) and mutant (red) profiles. (6) The percentage of beads with mutant DNA was determined by FACS and used in conjunction with the qPCR data to determine the minimum number of copies present to allow reliable detection of the mutant message.
    Turbo Dna Free Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/turbo dna free kit/product/Thermo Fisher
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    turbo dna free kit - by Bioz Stars, 2021-03
    97/100 stars
      Buy from Supplier

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    Overview of EV-BEAMing. EVs from serum or CSF samples were pelleted at 100,000 g for 80 minutes and processed as follows: (1) RNA was extracted and (2) analyzed for total yield and quality using the Bioanalyzer (Agilent). (3) RNA was then reverse transcribed into cDNA and 1/2 of the sample was used to determine the IDH1 cDNA copy number inside EVs by (4) qPCR analysis. (5) The remaining sample was preamplified (14 cycles) and used as input for BEAMing PCR. The resulting DNA-coated beads were interrogated with sequence-specific fluorescent probes to produce beads with wild-type (green) and mutant (red) profiles. (6) The percentage of beads with mutant DNA was determined by FACS and used in conjunction with the qPCR data to determine the minimum number of copies present to allow reliable detection of the mutant message.

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: BEAMing and Droplet Digital PCR Analysis of Mutant IDH1 mRNA in Glioma Patient Serum and Cerebrospinal Fluid Extracellular Vesicles

    doi: 10.1038/mtna.2013.28

    Figure Lengend Snippet: Overview of EV-BEAMing. EVs from serum or CSF samples were pelleted at 100,000 g for 80 minutes and processed as follows: (1) RNA was extracted and (2) analyzed for total yield and quality using the Bioanalyzer (Agilent). (3) RNA was then reverse transcribed into cDNA and 1/2 of the sample was used to determine the IDH1 cDNA copy number inside EVs by (4) qPCR analysis. (5) The remaining sample was preamplified (14 cycles) and used as input for BEAMing PCR. The resulting DNA-coated beads were interrogated with sequence-specific fluorescent probes to produce beads with wild-type (green) and mutant (red) profiles. (6) The percentage of beads with mutant DNA was determined by FACS and used in conjunction with the qPCR data to determine the minimum number of copies present to allow reliable detection of the mutant message.

    Article Snippet: External DNA on the EVs was removed using 2 µl of DNAse from the Turbo DNA-free kit (Ambion, Foster City, CA), according to manufacturer's recommendations.

    Techniques: Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Sequencing, Mutagenesis, FACS

    Expression of CES genes in opossum. Liver and intestinal cDNAs were reverse transcribed from DNase I-treated RNA, and they were used as templates in RT-PCR to analyze CES gene expression. Lanes 2 and 3 are RT-PCR products amplified from liver (L) and intestine (I) cDNAs for the CES1 gene; lanes 4 and 5, RT-PCR products from liver (L) and intestine (I) cDNAs for the CES2.1 gene; lanes 6 and 7, RT-PCR products from liver (L) and intestine (I) for cDNAs for the CES2.2 gene; and lanes 8 and 9, RT-PCR products from liver (L) and intestine (I) for cDNAs for the CES2.3 gene. M shows the DNA size ladder.

    Journal: BMC Evolutionary Biology

    Article Title: Opossum carboxylesterases: sequences, phylogeny and evidence for CES gene duplication events predating the marsupial-eutherian common ancestor

    doi: 10.1186/1471-2148-8-54

    Figure Lengend Snippet: Expression of CES genes in opossum. Liver and intestinal cDNAs were reverse transcribed from DNase I-treated RNA, and they were used as templates in RT-PCR to analyze CES gene expression. Lanes 2 and 3 are RT-PCR products amplified from liver (L) and intestine (I) cDNAs for the CES1 gene; lanes 4 and 5, RT-PCR products from liver (L) and intestine (I) cDNAs for the CES2.1 gene; lanes 6 and 7, RT-PCR products from liver (L) and intestine (I) for cDNAs for the CES2.2 gene; and lanes 8 and 9, RT-PCR products from liver (L) and intestine (I) for cDNAs for the CES2.3 gene. M shows the DNA size ladder.

    Article Snippet: RT-PCR Expression Studies and Sequencing of RT-PCR Products Total RNA was isolated from livers and small intestines of opossums using the TRI Reagent (Molecular Research Center, Cincinnati, OH), and treated with DNase I from the Turbo DNA-free kit (Applied Biosystems) according to the manufacturer's protocol to remove contaminating DNA from the RNA preparations.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Amplification

    HEATR2 splice mutation results in alteration of the final conserved HEAT repeat and protein instability. (A) Pedigree of related families of UK-Pakistani descent. IV∶4 identifies the proband, also designated by the arrow. Solid symbols (individuals IV∶1, IV∶4 and IV∶10) indicate those affected with PCD. Double lines indicate consanguineous marriages. The individuals labeled DNA signified those that had their DNA included in SNP genotyping. (B) Schematic of HEATR2 transcript showing the transversion mutation ( ENST00000297440:c.2432-1G > C ) affecting the splice acceptor site of the final exon. The mutation results in inactivation of this splice site and utilization of an adjacent downstream cryptic splice acceptor site in exon 13, causing a 2-nucleotide AG deletion in the HEATR2 transcript, resulting in a frameshift in translation ( Figure S2B ). This is predicted to alter the final 44 amino acids of the protein and add an additional 33 amino acids with creation of a novel termination signal at codon 888 in the 3′UTR (See Figure S3A ). This mutation disrupts the final highly conserved HEAT repeat and alters the C-terminus of the ARM-type fold superfamily domain (red). (C) Relative expression levels of HEATR2 transcript by RT-qPCR, when normalized to the reference TBP gene. (D) The PCD transversion mutation ( ENST00000297440:c.2432-1G > C ) does not affect HEATR2 transcript stability or gross splicing as shown by RT-PCR on parental control (C) and patient (P*) cDNA from LCLs. PCR products spanning the gene including the splice acceptor mutation at Exon 11–13 and Exon 12-3′UTR show no obvious alterations in size. Direct sequencing confirmed a 2 base pair deletion consistent with efficient splicing to the cryptic splice acceptor at the start of exon 13 in PCD patients ( Figure S2B ). (E) Western blot analysis on total protein extracts from unrelated control, heterozygous parental and homozygous patient LCLs demonstrates the PCD mutation ( ENST00000297440:c.2432-1G > C ) results in an elongated HEATR2 protein present at reduced levels implying instability. The slight shift in mobility of the protein in the patient is consistent with the predicted 3 kDa size shift due to the amino acid alterations described. β-actin is used as a loading control. (For longer exposure see Figure S3B ). (F) Levels of HEATR2 protein normalized relative to β-actin reveal that parental samples which are heterozygous for the mutation shows a reduction to ≈50% of that of unrelated controls whilst the homozygous patient sample shows a reduction to ≈3% of control levels.

    Journal: PLoS Genetics

    Article Title: HEATR2 Plays a Conserved Role in Assembly of the Ciliary Motile Apparatus

    doi: 10.1371/journal.pgen.1004577

    Figure Lengend Snippet: HEATR2 splice mutation results in alteration of the final conserved HEAT repeat and protein instability. (A) Pedigree of related families of UK-Pakistani descent. IV∶4 identifies the proband, also designated by the arrow. Solid symbols (individuals IV∶1, IV∶4 and IV∶10) indicate those affected with PCD. Double lines indicate consanguineous marriages. The individuals labeled DNA signified those that had their DNA included in SNP genotyping. (B) Schematic of HEATR2 transcript showing the transversion mutation ( ENST00000297440:c.2432-1G > C ) affecting the splice acceptor site of the final exon. The mutation results in inactivation of this splice site and utilization of an adjacent downstream cryptic splice acceptor site in exon 13, causing a 2-nucleotide AG deletion in the HEATR2 transcript, resulting in a frameshift in translation ( Figure S2B ). This is predicted to alter the final 44 amino acids of the protein and add an additional 33 amino acids with creation of a novel termination signal at codon 888 in the 3′UTR (See Figure S3A ). This mutation disrupts the final highly conserved HEAT repeat and alters the C-terminus of the ARM-type fold superfamily domain (red). (C) Relative expression levels of HEATR2 transcript by RT-qPCR, when normalized to the reference TBP gene. (D) The PCD transversion mutation ( ENST00000297440:c.2432-1G > C ) does not affect HEATR2 transcript stability or gross splicing as shown by RT-PCR on parental control (C) and patient (P*) cDNA from LCLs. PCR products spanning the gene including the splice acceptor mutation at Exon 11–13 and Exon 12-3′UTR show no obvious alterations in size. Direct sequencing confirmed a 2 base pair deletion consistent with efficient splicing to the cryptic splice acceptor at the start of exon 13 in PCD patients ( Figure S2B ). (E) Western blot analysis on total protein extracts from unrelated control, heterozygous parental and homozygous patient LCLs demonstrates the PCD mutation ( ENST00000297440:c.2432-1G > C ) results in an elongated HEATR2 protein present at reduced levels implying instability. The slight shift in mobility of the protein in the patient is consistent with the predicted 3 kDa size shift due to the amino acid alterations described. β-actin is used as a loading control. (For longer exposure see Figure S3B ). (F) Levels of HEATR2 protein normalized relative to β-actin reveal that parental samples which are heterozygous for the mutation shows a reduction to ≈50% of that of unrelated controls whilst the homozygous patient sample shows a reduction to ≈3% of control levels.

    Article Snippet: Total RNA was isolated according to manufacturer's protocol using RNAeasy minicolumns (Qiagen), followed by DNase treatment with Turbo DNA-free kit (Ambion). cDNA was made using First Strand Synthesis of cDNA for RT-PCR (AMV) kit (Roche).

    Techniques: Mutagenesis, Labeling, Expressing, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Sequencing, Western Blot

    CRISPR/Cas9-mediated deletion of Plasmodium yoelii sera1 gene. (A) Schematic construct for disrupting the Pysera1 gene. The plasmid contains Cas9 and sgRNA expression cassettes and donor template for HR repair after a double-strand break (DSB) at the 3′ end of the Pysera1 exon 2 (red thunderbolt). The DNA inserted (In) between the left and right arms was added to detect donor integration in the design of the PCR primers. Exons 1 to 4 are indicated by the yellow boxes. TS (blue box) indicates the sgRNA target sequence. The positions and directions of primers p10 to p21 are indicated by the small black arrows. (B) PCR analysis of 5′ and 3′ integrations in P . yoelii 17XNL parasite and plasmid-transfected uncloned cultures. The positions of primers (e.g., p10/p12) are shown in panel A. Ctrl, control. (C) PCR screening of clonal parasites for targeted Pysera1 deletion. DNAs from five individual clones (c1 to c5) and from 17XNL parasite were screened. (D) DNA sequencing confirms a 5.0-kb deletion in the Pysera1 gene from clonal parasite c1. The top panel shows the partial nucleotide sequence of the left and right arms from parental strain 17XNL. The bottom panel shows the 46-bp DNA insert between the left and right arm sequences in clone c1. (E) RT-PCR of Pysera1 mRNA from parental strain 17XNL and clones c1, c2, and c3, showing the lack of Pysera1 transcription in the three cloned lines. PCR amplification of the cDNA after reverse transcription (+) and PCR amplification of the RNA without reverse transcription (−) are indicated. P . yoelii gapdh mRNA serves as the endogenous control.

    Journal: mBio

    Article Title: Efficient Editing of Malaria Parasite Genome Using the CRISPR/Cas9 System

    doi: 10.1128/mBio.01414-14

    Figure Lengend Snippet: CRISPR/Cas9-mediated deletion of Plasmodium yoelii sera1 gene. (A) Schematic construct for disrupting the Pysera1 gene. The plasmid contains Cas9 and sgRNA expression cassettes and donor template for HR repair after a double-strand break (DSB) at the 3′ end of the Pysera1 exon 2 (red thunderbolt). The DNA inserted (In) between the left and right arms was added to detect donor integration in the design of the PCR primers. Exons 1 to 4 are indicated by the yellow boxes. TS (blue box) indicates the sgRNA target sequence. The positions and directions of primers p10 to p21 are indicated by the small black arrows. (B) PCR analysis of 5′ and 3′ integrations in P . yoelii 17XNL parasite and plasmid-transfected uncloned cultures. The positions of primers (e.g., p10/p12) are shown in panel A. Ctrl, control. (C) PCR screening of clonal parasites for targeted Pysera1 deletion. DNAs from five individual clones (c1 to c5) and from 17XNL parasite were screened. (D) DNA sequencing confirms a 5.0-kb deletion in the Pysera1 gene from clonal parasite c1. The top panel shows the partial nucleotide sequence of the left and right arms from parental strain 17XNL. The bottom panel shows the 46-bp DNA insert between the left and right arm sequences in clone c1. (E) RT-PCR of Pysera1 mRNA from parental strain 17XNL and clones c1, c2, and c3, showing the lack of Pysera1 transcription in the three cloned lines. PCR amplification of the cDNA after reverse transcription (+) and PCR amplification of the RNA without reverse transcription (−) are indicated. P . yoelii gapdh mRNA serves as the endogenous control.

    Article Snippet: Purified RNA was treated with DNase using Turbo DNA-free kit (Life Technologies). cDNA was synthesized using RevertAid reverse transcriptase (Fermentas).

    Techniques: CRISPR, Construct, Plasmid Preparation, Expressing, Polymerase Chain Reaction, Sequencing, Transfection, Clone Assay, DNA Sequencing, Reverse Transcription Polymerase Chain Reaction, Amplification