pcr clean up kit  (TaKaRa)

 
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    Name:
    LA PCR Kit
    Description:
    The LA PCR Kit Version 2 1 contains all the reagents needed for amplification of long DNA templates enabling routine amplification of products up to 20 kb in length including GC rich amplicons For some DNA templates amplification of up to 48 kb is possible This long range PCR kit contains TaKaRa LA Taq DNA Polymerase buffers MgCl2 dNTPs molecular weight markers and control templates plus corresponding primers to ensure optimal PCR performance during long PCR
    Catalog Number:
    rr013b
    Price:
    None
    Size:
    100 Rxns
    Category:
    LA Taq PCR kit LA Taq products Long range PCR PCR
    Buy from Supplier


    Structured Review

    TaKaRa pcr clean up kit
    Essential role of PRAMEF12 in spermatogonial differentiation. a Immunofluorescence of whole-mount testes from P2, P4, and P7 Pramef12 Null and Pramef12 Het mice after staining with antibodies to DDX4 (left) or KIT (middle) and merged (right). KIT-positive spermatogonia were rarely detected at P2 in either Pramef12 Null or Pramef12 Het tubules but were present at P4 and increased at P7 in Pramef12 Het tubules. Few KIT-positive spermatogonia (arrow) were observed in P7 Pramef12 Null tubules. Scale bar, 50 μm. b Immunohistochemistry of sections from P7 Pramef12 Null (top panels) and Pramef12 Het (bottom panels) testes after staining with antibodies to DDX4 (left) or KIT (middle) and merged with Hoechst 33342 to stain <t>DNA</t> (right). Arrowheads, KIT-positive cells in Pramef12 Null testes. Scale bar, 50 μm. c Quantitative real-time <t>RT-PCR</t> of Kit mRNA abundance in P7 Pramef12 Null and Pramef12 Het testes using β-actin as an internal load control and setting the abundance in Pramef12 Het testis to 1. Mean ± s.d, n = 3 biologically independent samples per condition. * P = 1.43E-05 by two-tailed Student’s t test. d Immunoblot of KIT protein in P7 Pramef12 Null and Pramef12 Het testes using α-tubulin as a load control. e Same as a , but with antibodies to SOHLH1, SOHLH2, and PLZF. The number of spermatogonia positive for SOHLH1 and SOHLH2 was significantly reduced in P7 Pramef12 Null tubules (top panels). Representative of n = 3 a , b , d , e independent biological replicates with similar results per condition
    The LA PCR Kit Version 2 1 contains all the reagents needed for amplification of long DNA templates enabling routine amplification of products up to 20 kb in length including GC rich amplicons For some DNA templates amplification of up to 48 kb is possible This long range PCR kit contains TaKaRa LA Taq DNA Polymerase buffers MgCl2 dNTPs molecular weight markers and control templates plus corresponding primers to ensure optimal PCR performance during long PCR
    https://www.bioz.com/result/pcr clean up kit/product/TaKaRa
    Average 99 stars, based on 92 article reviews
    Price from $9.99 to $1999.99
    pcr clean up kit - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "Sertoli cell-only phenotype and scRNA-seq define PRAMEF12 as a factor essential for spermatogenesis in mice"

    Article Title: Sertoli cell-only phenotype and scRNA-seq define PRAMEF12 as a factor essential for spermatogenesis in mice

    Journal: Nature Communications

    doi: 10.1038/s41467-019-13193-3

    Essential role of PRAMEF12 in spermatogonial differentiation. a Immunofluorescence of whole-mount testes from P2, P4, and P7 Pramef12 Null and Pramef12 Het mice after staining with antibodies to DDX4 (left) or KIT (middle) and merged (right). KIT-positive spermatogonia were rarely detected at P2 in either Pramef12 Null or Pramef12 Het tubules but were present at P4 and increased at P7 in Pramef12 Het tubules. Few KIT-positive spermatogonia (arrow) were observed in P7 Pramef12 Null tubules. Scale bar, 50 μm. b Immunohistochemistry of sections from P7 Pramef12 Null (top panels) and Pramef12 Het (bottom panels) testes after staining with antibodies to DDX4 (left) or KIT (middle) and merged with Hoechst 33342 to stain DNA (right). Arrowheads, KIT-positive cells in Pramef12 Null testes. Scale bar, 50 μm. c Quantitative real-time RT-PCR of Kit mRNA abundance in P7 Pramef12 Null and Pramef12 Het testes using β-actin as an internal load control and setting the abundance in Pramef12 Het testis to 1. Mean ± s.d, n = 3 biologically independent samples per condition. * P = 1.43E-05 by two-tailed Student’s t test. d Immunoblot of KIT protein in P7 Pramef12 Null and Pramef12 Het testes using α-tubulin as a load control. e Same as a , but with antibodies to SOHLH1, SOHLH2, and PLZF. The number of spermatogonia positive for SOHLH1 and SOHLH2 was significantly reduced in P7 Pramef12 Null tubules (top panels). Representative of n = 3 a , b , d , e independent biological replicates with similar results per condition
    Figure Legend Snippet: Essential role of PRAMEF12 in spermatogonial differentiation. a Immunofluorescence of whole-mount testes from P2, P4, and P7 Pramef12 Null and Pramef12 Het mice after staining with antibodies to DDX4 (left) or KIT (middle) and merged (right). KIT-positive spermatogonia were rarely detected at P2 in either Pramef12 Null or Pramef12 Het tubules but were present at P4 and increased at P7 in Pramef12 Het tubules. Few KIT-positive spermatogonia (arrow) were observed in P7 Pramef12 Null tubules. Scale bar, 50 μm. b Immunohistochemistry of sections from P7 Pramef12 Null (top panels) and Pramef12 Het (bottom panels) testes after staining with antibodies to DDX4 (left) or KIT (middle) and merged with Hoechst 33342 to stain DNA (right). Arrowheads, KIT-positive cells in Pramef12 Null testes. Scale bar, 50 μm. c Quantitative real-time RT-PCR of Kit mRNA abundance in P7 Pramef12 Null and Pramef12 Het testes using β-actin as an internal load control and setting the abundance in Pramef12 Het testis to 1. Mean ± s.d, n = 3 biologically independent samples per condition. * P = 1.43E-05 by two-tailed Student’s t test. d Immunoblot of KIT protein in P7 Pramef12 Null and Pramef12 Het testes using α-tubulin as a load control. e Same as a , but with antibodies to SOHLH1, SOHLH2, and PLZF. The number of spermatogonia positive for SOHLH1 and SOHLH2 was significantly reduced in P7 Pramef12 Null tubules (top panels). Representative of n = 3 a , b , d , e independent biological replicates with similar results per condition

    Techniques Used: Immunofluorescence, Mouse Assay, Staining, Immunohistochemistry, Quantitative RT-PCR, Two Tailed Test

    2) Product Images from "Microfluidic PCR Amplification and MiSeq Amplicon Sequencing Techniques for High-Throughput Detection and Genotyping of Human Pathogenic RNA Viruses in Human Feces, Sewage, and Oysters"

    Article Title: Microfluidic PCR Amplification and MiSeq Amplicon Sequencing Techniques for High-Throughput Detection and Genotyping of Human Pathogenic RNA Viruses in Human Feces, Sewage, and Oysters

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.00830

    Pipeline of microfluidic nested PCR followed by MiSeq amplicon sequencing (MFnPCR–MiSeq). Purification of PCR amplicons was carried out with the NucleoSpin Gel and PCR Clean-up Kit. Amplification by the first-round and nested PCR was performed on a 48.48 Access Array (AA) chip in a BioMark HD reader, while the third PCR for addition of a sample-specific tag and Illumina adapter was run in 96-well PCR plates on a conventional thermal cycler.
    Figure Legend Snippet: Pipeline of microfluidic nested PCR followed by MiSeq amplicon sequencing (MFnPCR–MiSeq). Purification of PCR amplicons was carried out with the NucleoSpin Gel and PCR Clean-up Kit. Amplification by the first-round and nested PCR was performed on a 48.48 Access Array (AA) chip in a BioMark HD reader, while the third PCR for addition of a sample-specific tag and Illumina adapter was run in 96-well PCR plates on a conventional thermal cycler.

    Techniques Used: Nested PCR, Amplification, Sequencing, Purification, Polymerase Chain Reaction, Chromatin Immunoprecipitation

    3) Product Images from "Microfluidic PCR Amplification and MiSeq Amplicon Sequencing Techniques for High-Throughput Detection and Genotyping of Human Pathogenic RNA Viruses in Human Feces, Sewage, and Oysters"

    Article Title: Microfluidic PCR Amplification and MiSeq Amplicon Sequencing Techniques for High-Throughput Detection and Genotyping of Human Pathogenic RNA Viruses in Human Feces, Sewage, and Oysters

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.00830

    Pipeline of microfluidic nested PCR followed by MiSeq amplicon sequencing (MFnPCR–MiSeq). Purification of PCR amplicons was carried out with the NucleoSpin Gel and PCR Clean-up Kit. Amplification by the first-round and nested PCR was performed on a 48.48 Access Array (AA) chip in a BioMark HD reader, while the third PCR for addition of a sample-specific tag and Illumina adapter was run in 96-well PCR plates on a conventional thermal cycler.
    Figure Legend Snippet: Pipeline of microfluidic nested PCR followed by MiSeq amplicon sequencing (MFnPCR–MiSeq). Purification of PCR amplicons was carried out with the NucleoSpin Gel and PCR Clean-up Kit. Amplification by the first-round and nested PCR was performed on a 48.48 Access Array (AA) chip in a BioMark HD reader, while the third PCR for addition of a sample-specific tag and Illumina adapter was run in 96-well PCR plates on a conventional thermal cycler.

    Techniques Used: Nested PCR, Amplification, Sequencing, Purification, Polymerase Chain Reaction, Chromatin Immunoprecipitation

    4) Product Images from "The “speed gene” effect of myostatin arises in Thoroughbred horses due to a promoter proximal SINE insertion"

    Article Title: The “speed gene” effect of myostatin arises in Thoroughbred horses due to a promoter proximal SINE insertion

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0205664

    The MSTN transcription start site is altered by the SINE insertion. RNA was isolated from equine gluteus medius skeletal muscle tissue and 5'RACE was used to recover sequence data for the 5'-end of the myostatin mRNA transcript. DNA was sequenced using M13 primers by MWG eurofins. (A) Displays snapshots of the raw sequencing data obtained with transcription start sites (TSS) indicated. The SMARTer 5'RACE primer sequence is shown prior to the TSS along with 8 additional bases (*) which were added to first-strand cDNA; during reverse transcription, when the SMARTScribe reverse transcriptase reaches the 5’ end of the RNA, its terminal transferase activity adds a few additional nucleotides to the 3’ end of the first-strand cDNA. The same set of 8 bases ( ACATGGGG ) is observed in each clone. (B) Depiction of a non-SINE insertion MSTN gene (top) and a SINE insertion MSTN gene (bottom), with experimentally determined TSS indicated. Red lines indicate the position of the SINE insertion sequence. Numbering of nucleotide bases established from the human TSS (and the in silico predicted equine transcription start site) as +1. Position of the ATG (predicted translation start site) and predicted TATA box are also marked on diagram. (C) 5'RACE PCR products were electrophoresed on a 1.5% agarose gel, 1: TT/NN non-SINE insertion sample; 2: CC/II SINE insertion sample; M1: wide range MW markers (Sigma).
    Figure Legend Snippet: The MSTN transcription start site is altered by the SINE insertion. RNA was isolated from equine gluteus medius skeletal muscle tissue and 5'RACE was used to recover sequence data for the 5'-end of the myostatin mRNA transcript. DNA was sequenced using M13 primers by MWG eurofins. (A) Displays snapshots of the raw sequencing data obtained with transcription start sites (TSS) indicated. The SMARTer 5'RACE primer sequence is shown prior to the TSS along with 8 additional bases (*) which were added to first-strand cDNA; during reverse transcription, when the SMARTScribe reverse transcriptase reaches the 5’ end of the RNA, its terminal transferase activity adds a few additional nucleotides to the 3’ end of the first-strand cDNA. The same set of 8 bases ( ACATGGGG ) is observed in each clone. (B) Depiction of a non-SINE insertion MSTN gene (top) and a SINE insertion MSTN gene (bottom), with experimentally determined TSS indicated. Red lines indicate the position of the SINE insertion sequence. Numbering of nucleotide bases established from the human TSS (and the in silico predicted equine transcription start site) as +1. Position of the ATG (predicted translation start site) and predicted TATA box are also marked on diagram. (C) 5'RACE PCR products were electrophoresed on a 1.5% agarose gel, 1: TT/NN non-SINE insertion sample; 2: CC/II SINE insertion sample; M1: wide range MW markers (Sigma).

    Techniques Used: Isolation, Sequencing, Activity Assay, In Silico, Polymerase Chain Reaction, Agarose Gel Electrophoresis

    5) Product Images from "The “speed gene” effect of myostatin arises in Thoroughbred horses due to a promoter proximal SINE insertion"

    Article Title: The “speed gene” effect of myostatin arises in Thoroughbred horses due to a promoter proximal SINE insertion

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0205664

    The MSTN transcription start site is altered by the SINE insertion. RNA was isolated from equine gluteus medius skeletal muscle tissue and 5'RACE was used to recover sequence data for the 5'-end of the myostatin mRNA transcript. DNA was sequenced using M13 primers by MWG eurofins. (A) Displays snapshots of the raw sequencing data obtained with transcription start sites (TSS) indicated. The SMARTer 5'RACE primer sequence is shown prior to the TSS along with 8 additional bases (*) which were added to first-strand cDNA; during reverse transcription, when the SMARTScribe reverse transcriptase reaches the 5’ end of the RNA, its terminal transferase activity adds a few additional nucleotides to the 3’ end of the first-strand cDNA. The same set of 8 bases ( ACATGGGG ) is observed in each clone. (B) Depiction of a non-SINE insertion MSTN gene (top) and a SINE insertion MSTN gene (bottom), with experimentally determined TSS indicated. Red lines indicate the position of the SINE insertion sequence. Numbering of nucleotide bases established from the human TSS (and the in silico predicted equine transcription start site) as +1. Position of the ATG (predicted translation start site) and predicted TATA box are also marked on diagram. (C) 5'RACE PCR products were electrophoresed on a 1.5% agarose gel, 1: TT/NN non-SINE insertion sample; 2: CC/II SINE insertion sample; M1: wide range MW markers (Sigma).
    Figure Legend Snippet: The MSTN transcription start site is altered by the SINE insertion. RNA was isolated from equine gluteus medius skeletal muscle tissue and 5'RACE was used to recover sequence data for the 5'-end of the myostatin mRNA transcript. DNA was sequenced using M13 primers by MWG eurofins. (A) Displays snapshots of the raw sequencing data obtained with transcription start sites (TSS) indicated. The SMARTer 5'RACE primer sequence is shown prior to the TSS along with 8 additional bases (*) which were added to first-strand cDNA; during reverse transcription, when the SMARTScribe reverse transcriptase reaches the 5’ end of the RNA, its terminal transferase activity adds a few additional nucleotides to the 3’ end of the first-strand cDNA. The same set of 8 bases ( ACATGGGG ) is observed in each clone. (B) Depiction of a non-SINE insertion MSTN gene (top) and a SINE insertion MSTN gene (bottom), with experimentally determined TSS indicated. Red lines indicate the position of the SINE insertion sequence. Numbering of nucleotide bases established from the human TSS (and the in silico predicted equine transcription start site) as +1. Position of the ATG (predicted translation start site) and predicted TATA box are also marked on diagram. (C) 5'RACE PCR products were electrophoresed on a 1.5% agarose gel, 1: TT/NN non-SINE insertion sample; 2: CC/II SINE insertion sample; M1: wide range MW markers (Sigma).

    Techniques Used: Isolation, Sequencing, Activity Assay, In Silico, Polymerase Chain Reaction, Agarose Gel Electrophoresis

    6) Product Images from "Dominant prevalence of Porphyromonas gingivalis fimA types I and IV in healthy Japanese children"

    Article Title: Dominant prevalence of Porphyromonas gingivalis fimA types I and IV in healthy Japanese children

    Journal: Journal of Dental Sciences

    doi: 10.1016/j.jds.2017.03.006

    Determination of fimA types by PCR . (A) PCR was performed with a set of fimA- universal primers. Lanes 1 to 5: 0.1, 1, 10, 100, and 1,000 pg DNA from P. gingivalis ATCC 33277, respectively; lanes 6 to 11: 5 ng DNA from ATCC 33277 (type I), 16-1 (type Ib), HW24D1 (type II), ATCC 49417 (type III), W83 (type IV), and HNA99 (type V), respectively. (B) PCR was performed with a set of fimA type-specific primers. Lanes 1–5: 0.1, 1, 10, 100, and 1,000 pg of corresponding DNA, respectively; lanes 6 to 11; 5 ng of DNA from types I, Ib, II, III, IV, and V, respectively.
    Figure Legend Snippet: Determination of fimA types by PCR . (A) PCR was performed with a set of fimA- universal primers. Lanes 1 to 5: 0.1, 1, 10, 100, and 1,000 pg DNA from P. gingivalis ATCC 33277, respectively; lanes 6 to 11: 5 ng DNA from ATCC 33277 (type I), 16-1 (type Ib), HW24D1 (type II), ATCC 49417 (type III), W83 (type IV), and HNA99 (type V), respectively. (B) PCR was performed with a set of fimA type-specific primers. Lanes 1–5: 0.1, 1, 10, 100, and 1,000 pg of corresponding DNA, respectively; lanes 6 to 11; 5 ng of DNA from types I, Ib, II, III, IV, and V, respectively.

    Techniques Used: Polymerase Chain Reaction

    7) Product Images from "Biotinylation of Deoxyguanosine at the Abasic Site in Double-Stranded Oligodeoxynucleotides"

    Article Title: Biotinylation of Deoxyguanosine at the Abasic Site in Double-Stranded Oligodeoxynucleotides

    Journal: Journal of Analytical Methods in Chemistry

    doi: 10.1155/2016/4681421

    Restriction digestion and gel electrophoresis of the PCR amplified products using SS-G (a), SS-abasic site (b), or the biotinylated DS-G/abasic site (c) as the template. The restriction enzyme MspI which recognizes the CCGG sequence was used to cleave the PCR amplified product.
    Figure Legend Snippet: Restriction digestion and gel electrophoresis of the PCR amplified products using SS-G (a), SS-abasic site (b), or the biotinylated DS-G/abasic site (c) as the template. The restriction enzyme MspI which recognizes the CCGG sequence was used to cleave the PCR amplified product.

    Techniques Used: Nucleic Acid Electrophoresis, Polymerase Chain Reaction, Amplification, Sequencing

    8) Product Images from "Microfluidic PCR Amplification and MiSeq Amplicon Sequencing Techniques for High-Throughput Detection and Genotyping of Human Pathogenic RNA Viruses in Human Feces, Sewage, and Oysters"

    Article Title: Microfluidic PCR Amplification and MiSeq Amplicon Sequencing Techniques for High-Throughput Detection and Genotyping of Human Pathogenic RNA Viruses in Human Feces, Sewage, and Oysters

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.00830

    Pipeline of microfluidic nested PCR followed by MiSeq amplicon sequencing (MFnPCR–MiSeq). Purification of PCR amplicons was carried out with the NucleoSpin Gel and PCR Clean-up Kit. Amplification by the first-round and nested PCR was performed on a 48.48 Access Array (AA) chip in a BioMark HD reader, while the third PCR for addition of a sample-specific tag and Illumina adapter was run in 96-well PCR plates on a conventional thermal cycler.
    Figure Legend Snippet: Pipeline of microfluidic nested PCR followed by MiSeq amplicon sequencing (MFnPCR–MiSeq). Purification of PCR amplicons was carried out with the NucleoSpin Gel and PCR Clean-up Kit. Amplification by the first-round and nested PCR was performed on a 48.48 Access Array (AA) chip in a BioMark HD reader, while the third PCR for addition of a sample-specific tag and Illumina adapter was run in 96-well PCR plates on a conventional thermal cycler.

    Techniques Used: Nested PCR, Amplification, Sequencing, Purification, Polymerase Chain Reaction, Chromatin Immunoprecipitation

    9) Product Images from "CRISPR/Cas9 knockout of HAS2 in rat chondrosarcoma chondrocytes demonstrates the requirement of hyaluronan for aggrecan retention"

    Article Title: CRISPR/Cas9 knockout of HAS2 in rat chondrosarcoma chondrocytes demonstrates the requirement of hyaluronan for aggrecan retention

    Journal: Matrix biology : journal of the International Society for Matrix Biology

    doi: 10.1016/j.matbio.2016.04.002

    Detection and determination of Has2 gene mutation A 454 bp region within exon2 of rat Has2 that contains the Cas9 mutation site was PCR amplified from genomic DNA of each cell clone as a template. The amplified product was gel-purified and then digested with Pas1. The final products were analyzed by agarose gel electrophoresis. DNA standards (std) are labeled. Panel A. Products amplified from RCS-o WT cells and clones 1 thru 7. Panel B. Pas1 digestion products of the amplified 454 bp products shown in panel A. Panel C. Products amplified from RCS-Cas9 WT cells and clones 3, 7, 39, 43, 53, 80 and 88. Panel D. Pas1 digestion products of the amplified 454 bp products shown in panel C. Panel E. Alignment of the targeted rat Has2 genomic sequences from RCS-Cas9 clone #7, allele 1 and allele 2 with the WT RCS-Cas9 cells. Deleted bases are indicated.
    Figure Legend Snippet: Detection and determination of Has2 gene mutation A 454 bp region within exon2 of rat Has2 that contains the Cas9 mutation site was PCR amplified from genomic DNA of each cell clone as a template. The amplified product was gel-purified and then digested with Pas1. The final products were analyzed by agarose gel electrophoresis. DNA standards (std) are labeled. Panel A. Products amplified from RCS-o WT cells and clones 1 thru 7. Panel B. Pas1 digestion products of the amplified 454 bp products shown in panel A. Panel C. Products amplified from RCS-Cas9 WT cells and clones 3, 7, 39, 43, 53, 80 and 88. Panel D. Pas1 digestion products of the amplified 454 bp products shown in panel C. Panel E. Alignment of the targeted rat Has2 genomic sequences from RCS-Cas9 clone #7, allele 1 and allele 2 with the WT RCS-Cas9 cells. Deleted bases are indicated.

    Techniques Used: Mutagenesis, Polymerase Chain Reaction, Amplification, Purification, Agarose Gel Electrophoresis, Labeling, Clone Assay, Genomic Sequencing

    Related Articles

    Amplification:

    Article Title: Murine lymph node-derived stromal cells effectively support survival but induce no activation/proliferation of peripheral resting T cells in vitro
    Article Snippet: .. Single-stranded cDNA was synthesized by reverse transcriptase (RT) with random hexamer oligonucleotides as primers, and the desired cDNA was amplified by polymerase chain reaction (PCR) using a TaKaRa RNA LA PCR kit (TaKaRa Biomedicals, Kyoto, Japan). .. Primers (sense and antisense) for the amplification of each cytokine cDNA were as follows: β-actin sense, TGG AAT CCT GTG GCA TCC ATG AAA C; β-actin antisense, TAA AAC GCA GCT CAG TAA CAG TCC G; IL-1α sense, CTC TAG AGC ACC ATG CTA CAG AC; IL-1α antisense, TGG AAT CCA GGG GAA ACA CTG; IL-2 sense, TGA TGG ACC TAC AGG AGC TCC TGA G; IL-2 antisense, GAG TCA AAT CCA GAA CAT GCC GCA G; IL-3 sense, GAA GTG GAT CCT GAG GAC AGA TAC G; IL-3 antisense, GAC CAT GGG CCA TGA GGA ACA TTC; IL-4 sense, CGA AGA ACA CCA CAG AGA GTG AGC T; IL-4 antisense, GAC TCA TTC ATG GTG CAG CTT ATC G; IL-5 sense, CTC TAG TAA GCC CAC TTC TA; IL-5 antisense, TGA TAC ATG AAT AAC ATC CC; IL-6 sense, TGG AGT CAC AGA AGG AGT GGC TAA G; IL-6 antisense, TCT GAC CAC AGA AGG AGT GGC TAA G; IL-7 sense, AAA TGC AGC TGA CTG CTG GC; IL-7 antisense, TCT CCA GTC TAA AAC AGG AC; IL-10 sense, TAC CTG GTA GAA GTG ATG CC; IL-10 antisense, CAT CAT GTA TGC TTC TAT GC; tumour necrosis factor-α (TNF-α) sense, GGC AGG TCT ACT TTG GAG TCA TTG C; TNF-α antisense, ACA TTC GAG GCT CCA GTG AAT TCG G; lymphotoxin (LT)-α sense, TGG CTG GGA ACA GGG GAA GGT TGA C; LT-α antisense, CGT GCT TTC TTC TAG AAC CCC TTG G. The conditions for PCR were 1 min at 94°, 2 min at 55° and 3 min at 72° for 30 cycles.

    Article Title: B-cell translocation gene 2 mediates crosstalk between PI3K/Akt1 and NF?B pathways which enhances transcription of MnSOD by accelerating I?B? degradation in normal and cancer cells
    Article Snippet: .. RT-PCR Total cellular RNAs (1.0 μg) isolated with RNAiso Plus were used for cDNA preparation and then amplified by PCR kit (Takara Inc., Japan); First strand cDNA was synthesized using oligo-dT by reverse transcription reaction in 10 μl of reaction volume. .. The gene of interest was amplified by ExTaq polymerase in PCR kits using primer sequences described in the Additional file .

    Article Title: Tomato yellow leaf curl virus intergenic siRNAs target a host long noncoding RNA to modulate disease symptoms
    Article Snippet: .. The 5’ flanking region of the sense transcripts of SlLNR1 were obtained by RNA ligase-mediated rapid amplification of 5' cDNA ends First Choice ® RLM-RACE Kit (Invitrogen, USA), according to the instructions of the manufacturer, and the 3’ end was verified by 3’ RACE PCR kit (TAKARA). .. Full sense transcript of the SlLNR1 was amplified with the primers designed according to the joint sequence by RT-PCR.

    Article Title: Cloning and characterization of an Eimeria necatrix gene encoding a gametocyte protein and associated with oocyst wall formation
    Article Snippet: .. The sequence of the gene coding the gametocyte protein was amplified by reverse transcription polymerase chain reaction (RT-PCR) using the RNA LA PCR Kit (TaKaRa Bio. ..

    Synthesized:

    Article Title: Murine lymph node-derived stromal cells effectively support survival but induce no activation/proliferation of peripheral resting T cells in vitro
    Article Snippet: .. Single-stranded cDNA was synthesized by reverse transcriptase (RT) with random hexamer oligonucleotides as primers, and the desired cDNA was amplified by polymerase chain reaction (PCR) using a TaKaRa RNA LA PCR kit (TaKaRa Biomedicals, Kyoto, Japan). .. Primers (sense and antisense) for the amplification of each cytokine cDNA were as follows: β-actin sense, TGG AAT CCT GTG GCA TCC ATG AAA C; β-actin antisense, TAA AAC GCA GCT CAG TAA CAG TCC G; IL-1α sense, CTC TAG AGC ACC ATG CTA CAG AC; IL-1α antisense, TGG AAT CCA GGG GAA ACA CTG; IL-2 sense, TGA TGG ACC TAC AGG AGC TCC TGA G; IL-2 antisense, GAG TCA AAT CCA GAA CAT GCC GCA G; IL-3 sense, GAA GTG GAT CCT GAG GAC AGA TAC G; IL-3 antisense, GAC CAT GGG CCA TGA GGA ACA TTC; IL-4 sense, CGA AGA ACA CCA CAG AGA GTG AGC T; IL-4 antisense, GAC TCA TTC ATG GTG CAG CTT ATC G; IL-5 sense, CTC TAG TAA GCC CAC TTC TA; IL-5 antisense, TGA TAC ATG AAT AAC ATC CC; IL-6 sense, TGG AGT CAC AGA AGG AGT GGC TAA G; IL-6 antisense, TCT GAC CAC AGA AGG AGT GGC TAA G; IL-7 sense, AAA TGC AGC TGA CTG CTG GC; IL-7 antisense, TCT CCA GTC TAA AAC AGG AC; IL-10 sense, TAC CTG GTA GAA GTG ATG CC; IL-10 antisense, CAT CAT GTA TGC TTC TAT GC; tumour necrosis factor-α (TNF-α) sense, GGC AGG TCT ACT TTG GAG TCA TTG C; TNF-α antisense, ACA TTC GAG GCT CCA GTG AAT TCG G; lymphotoxin (LT)-α sense, TGG CTG GGA ACA GGG GAA GGT TGA C; LT-α antisense, CGT GCT TTC TTC TAG AAC CCC TTG G. The conditions for PCR were 1 min at 94°, 2 min at 55° and 3 min at 72° for 30 cycles.

    Article Title: B-cell translocation gene 2 mediates crosstalk between PI3K/Akt1 and NF?B pathways which enhances transcription of MnSOD by accelerating I?B? degradation in normal and cancer cells
    Article Snippet: .. RT-PCR Total cellular RNAs (1.0 μg) isolated with RNAiso Plus were used for cDNA preparation and then amplified by PCR kit (Takara Inc., Japan); First strand cDNA was synthesized using oligo-dT by reverse transcription reaction in 10 μl of reaction volume. .. The gene of interest was amplified by ExTaq polymerase in PCR kits using primer sequences described in the Additional file .

    Isolation:

    Article Title: B-cell translocation gene 2 mediates crosstalk between PI3K/Akt1 and NF?B pathways which enhances transcription of MnSOD by accelerating I?B? degradation in normal and cancer cells
    Article Snippet: .. RT-PCR Total cellular RNAs (1.0 μg) isolated with RNAiso Plus were used for cDNA preparation and then amplified by PCR kit (Takara Inc., Japan); First strand cDNA was synthesized using oligo-dT by reverse transcription reaction in 10 μl of reaction volume. .. The gene of interest was amplified by ExTaq polymerase in PCR kits using primer sequences described in the Additional file .

    Produced:

    Article Title: The Bmdsx transgene including trimmed introns is sex-specifically spliced in tissues of the silkworm, Bombyx mori
    Article Snippet: .. RT-PCR was performed using the LA RNA PCR kit (Takara, www.takara-bio.co.jp ) following the manufacturer's instructions. cDNA was produced by random priming. .. RT-PCR primers were as follows: endogenous BmA3 , BmA3QPCR1F (5′-TACAATGAGCTGCGTGTCG-3′) and BmA3QPCR1R (5′-CGGGCGTGTTGAATGTTTC -3′); and Bmdsx mRNA transcribed from the transgene, TGM2F (5′-ATTGGCGGGACACGATC-3′) and TGM2R (5′-AGCGCTCCGTAGCACAA-3′).

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: B-cell translocation gene 2 mediates crosstalk between PI3K/Akt1 and NF?B pathways which enhances transcription of MnSOD by accelerating I?B? degradation in normal and cancer cells
    Article Snippet: .. RT-PCR Total cellular RNAs (1.0 μg) isolated with RNAiso Plus were used for cDNA preparation and then amplified by PCR kit (Takara Inc., Japan); First strand cDNA was synthesized using oligo-dT by reverse transcription reaction in 10 μl of reaction volume. .. The gene of interest was amplified by ExTaq polymerase in PCR kits using primer sequences described in the Additional file .

    Article Title: The Bmdsx transgene including trimmed introns is sex-specifically spliced in tissues of the silkworm, Bombyx mori
    Article Snippet: .. RT-PCR was performed using the LA RNA PCR kit (Takara, www.takara-bio.co.jp ) following the manufacturer's instructions. cDNA was produced by random priming. .. RT-PCR primers were as follows: endogenous BmA3 , BmA3QPCR1F (5′-TACAATGAGCTGCGTGTCG-3′) and BmA3QPCR1R (5′-CGGGCGTGTTGAATGTTTC -3′); and Bmdsx mRNA transcribed from the transgene, TGM2F (5′-ATTGGCGGGACACGATC-3′) and TGM2R (5′-AGCGCTCCGTAGCACAA-3′).

    Article Title: Cloning and characterization of an Eimeria necatrix gene encoding a gametocyte protein and associated with oocyst wall formation
    Article Snippet: .. The sequence of the gene coding the gametocyte protein was amplified by reverse transcription polymerase chain reaction (RT-PCR) using the RNA LA PCR Kit (TaKaRa Bio. ..

    Article Title: Spermidine Synthase Genes Are Essential for Survival of Arabidopsis
    Article Snippet: .. RT-PCR was conducted by using the RNA LA PCR Kit (Takara, Kyoto) with 0.5 μ g of total RNA. ..

    Random Hexamer Labeling:

    Article Title: Murine lymph node-derived stromal cells effectively support survival but induce no activation/proliferation of peripheral resting T cells in vitro
    Article Snippet: .. Single-stranded cDNA was synthesized by reverse transcriptase (RT) with random hexamer oligonucleotides as primers, and the desired cDNA was amplified by polymerase chain reaction (PCR) using a TaKaRa RNA LA PCR kit (TaKaRa Biomedicals, Kyoto, Japan). .. Primers (sense and antisense) for the amplification of each cytokine cDNA were as follows: β-actin sense, TGG AAT CCT GTG GCA TCC ATG AAA C; β-actin antisense, TAA AAC GCA GCT CAG TAA CAG TCC G; IL-1α sense, CTC TAG AGC ACC ATG CTA CAG AC; IL-1α antisense, TGG AAT CCA GGG GAA ACA CTG; IL-2 sense, TGA TGG ACC TAC AGG AGC TCC TGA G; IL-2 antisense, GAG TCA AAT CCA GAA CAT GCC GCA G; IL-3 sense, GAA GTG GAT CCT GAG GAC AGA TAC G; IL-3 antisense, GAC CAT GGG CCA TGA GGA ACA TTC; IL-4 sense, CGA AGA ACA CCA CAG AGA GTG AGC T; IL-4 antisense, GAC TCA TTC ATG GTG CAG CTT ATC G; IL-5 sense, CTC TAG TAA GCC CAC TTC TA; IL-5 antisense, TGA TAC ATG AAT AAC ATC CC; IL-6 sense, TGG AGT CAC AGA AGG AGT GGC TAA G; IL-6 antisense, TCT GAC CAC AGA AGG AGT GGC TAA G; IL-7 sense, AAA TGC AGC TGA CTG CTG GC; IL-7 antisense, TCT CCA GTC TAA AAC AGG AC; IL-10 sense, TAC CTG GTA GAA GTG ATG CC; IL-10 antisense, CAT CAT GTA TGC TTC TAT GC; tumour necrosis factor-α (TNF-α) sense, GGC AGG TCT ACT TTG GAG TCA TTG C; TNF-α antisense, ACA TTC GAG GCT CCA GTG AAT TCG G; lymphotoxin (LT)-α sense, TGG CTG GGA ACA GGG GAA GGT TGA C; LT-α antisense, CGT GCT TTC TTC TAG AAC CCC TTG G. The conditions for PCR were 1 min at 94°, 2 min at 55° and 3 min at 72° for 30 cycles.

    Polymerase Chain Reaction:

    Article Title: Murine lymph node-derived stromal cells effectively support survival but induce no activation/proliferation of peripheral resting T cells in vitro
    Article Snippet: .. Single-stranded cDNA was synthesized by reverse transcriptase (RT) with random hexamer oligonucleotides as primers, and the desired cDNA was amplified by polymerase chain reaction (PCR) using a TaKaRa RNA LA PCR kit (TaKaRa Biomedicals, Kyoto, Japan). .. Primers (sense and antisense) for the amplification of each cytokine cDNA were as follows: β-actin sense, TGG AAT CCT GTG GCA TCC ATG AAA C; β-actin antisense, TAA AAC GCA GCT CAG TAA CAG TCC G; IL-1α sense, CTC TAG AGC ACC ATG CTA CAG AC; IL-1α antisense, TGG AAT CCA GGG GAA ACA CTG; IL-2 sense, TGA TGG ACC TAC AGG AGC TCC TGA G; IL-2 antisense, GAG TCA AAT CCA GAA CAT GCC GCA G; IL-3 sense, GAA GTG GAT CCT GAG GAC AGA TAC G; IL-3 antisense, GAC CAT GGG CCA TGA GGA ACA TTC; IL-4 sense, CGA AGA ACA CCA CAG AGA GTG AGC T; IL-4 antisense, GAC TCA TTC ATG GTG CAG CTT ATC G; IL-5 sense, CTC TAG TAA GCC CAC TTC TA; IL-5 antisense, TGA TAC ATG AAT AAC ATC CC; IL-6 sense, TGG AGT CAC AGA AGG AGT GGC TAA G; IL-6 antisense, TCT GAC CAC AGA AGG AGT GGC TAA G; IL-7 sense, AAA TGC AGC TGA CTG CTG GC; IL-7 antisense, TCT CCA GTC TAA AAC AGG AC; IL-10 sense, TAC CTG GTA GAA GTG ATG CC; IL-10 antisense, CAT CAT GTA TGC TTC TAT GC; tumour necrosis factor-α (TNF-α) sense, GGC AGG TCT ACT TTG GAG TCA TTG C; TNF-α antisense, ACA TTC GAG GCT CCA GTG AAT TCG G; lymphotoxin (LT)-α sense, TGG CTG GGA ACA GGG GAA GGT TGA C; LT-α antisense, CGT GCT TTC TTC TAG AAC CCC TTG G. The conditions for PCR were 1 min at 94°, 2 min at 55° and 3 min at 72° for 30 cycles.

    Article Title: B-cell translocation gene 2 mediates crosstalk between PI3K/Akt1 and NF?B pathways which enhances transcription of MnSOD by accelerating I?B? degradation in normal and cancer cells
    Article Snippet: .. RT-PCR Total cellular RNAs (1.0 μg) isolated with RNAiso Plus were used for cDNA preparation and then amplified by PCR kit (Takara Inc., Japan); First strand cDNA was synthesized using oligo-dT by reverse transcription reaction in 10 μl of reaction volume. .. The gene of interest was amplified by ExTaq polymerase in PCR kits using primer sequences described in the Additional file .

    Article Title: The Riemerella anatipestifer M949_RS01035 gene is involved in bacterial lipopolysaccharide biosynthesis
    Article Snippet: .. Primer pairs specific for Tn4351 (primers TN-1 and IS4351-F) were used to amplify the sequences adjacent to the insertion site using the LA PCR kit (TaKaRa Biotechnology (Dalian) Co., Ltd., Dalian, China). .. The nucleotide sequence was compared to sequence in the National Center for Biotechnology Information database using the BLASTX program [ ].

    Article Title: The Bmdsx transgene including trimmed introns is sex-specifically spliced in tissues of the silkworm, Bombyx mori
    Article Snippet: .. RT-PCR was performed using the LA RNA PCR kit (Takara, www.takara-bio.co.jp ) following the manufacturer's instructions. cDNA was produced by random priming. .. RT-PCR primers were as follows: endogenous BmA3 , BmA3QPCR1F (5′-TACAATGAGCTGCGTGTCG-3′) and BmA3QPCR1R (5′-CGGGCGTGTTGAATGTTTC -3′); and Bmdsx mRNA transcribed from the transgene, TGM2F (5′-ATTGGCGGGACACGATC-3′) and TGM2R (5′-AGCGCTCCGTAGCACAA-3′).

    Article Title: Tomato yellow leaf curl virus intergenic siRNAs target a host long noncoding RNA to modulate disease symptoms
    Article Snippet: .. The 5’ flanking region of the sense transcripts of SlLNR1 were obtained by RNA ligase-mediated rapid amplification of 5' cDNA ends First Choice ® RLM-RACE Kit (Invitrogen, USA), according to the instructions of the manufacturer, and the 3’ end was verified by 3’ RACE PCR kit (TAKARA). .. Full sense transcript of the SlLNR1 was amplified with the primers designed according to the joint sequence by RT-PCR.

    Article Title: Cloning and characterization of an Eimeria necatrix gene encoding a gametocyte protein and associated with oocyst wall formation
    Article Snippet: .. The sequence of the gene coding the gametocyte protein was amplified by reverse transcription polymerase chain reaction (RT-PCR) using the RNA LA PCR Kit (TaKaRa Bio. ..

    Article Title: Spermidine Synthase Genes Are Essential for Survival of Arabidopsis
    Article Snippet: .. RT-PCR was conducted by using the RNA LA PCR Kit (Takara, Kyoto) with 0.5 μ g of total RNA. ..

    Article Title: Characterization of a novel germline PALB2 duplication in a hereditary breast and ovarian cancer family
    Article Snippet: .. LR-PCR was performed using a forward primer in intron 12 and a reverse primer in 3′-untranslated region (UTR) of PALB2 and the TaKaRa LA PCR kit (TaKaRa, Clontech) following the manufacturer’s instructions. ..

    Sequencing:

    Article Title: Cloning and characterization of an Eimeria necatrix gene encoding a gametocyte protein and associated with oocyst wall formation
    Article Snippet: .. The sequence of the gene coding the gametocyte protein was amplified by reverse transcription polymerase chain reaction (RT-PCR) using the RNA LA PCR Kit (TaKaRa Bio. ..

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  • 99
    TaKaRa pcr clean up kit
    Essential role of PRAMEF12 in spermatogonial differentiation. a Immunofluorescence of whole-mount testes from P2, P4, and P7 Pramef12 Null and Pramef12 Het mice after staining with antibodies to DDX4 (left) or KIT (middle) and merged (right). KIT-positive spermatogonia were rarely detected at P2 in either Pramef12 Null or Pramef12 Het tubules but were present at P4 and increased at P7 in Pramef12 Het tubules. Few KIT-positive spermatogonia (arrow) were observed in P7 Pramef12 Null tubules. Scale bar, 50 μm. b Immunohistochemistry of sections from P7 Pramef12 Null (top panels) and Pramef12 Het (bottom panels) testes after staining with antibodies to DDX4 (left) or KIT (middle) and merged with Hoechst 33342 to stain <t>DNA</t> (right). Arrowheads, KIT-positive cells in Pramef12 Null testes. Scale bar, 50 μm. c Quantitative real-time <t>RT-PCR</t> of Kit mRNA abundance in P7 Pramef12 Null and Pramef12 Het testes using β-actin as an internal load control and setting the abundance in Pramef12 Het testis to 1. Mean ± s.d, n = 3 biologically independent samples per condition. * P = 1.43E-05 by two-tailed Student’s t test. d Immunoblot of KIT protein in P7 Pramef12 Null and Pramef12 Het testes using α-tubulin as a load control. e Same as a , but with antibodies to SOHLH1, SOHLH2, and PLZF. The number of spermatogonia positive for SOHLH1 and SOHLH2 was significantly reduced in P7 Pramef12 Null tubules (top panels). Representative of n = 3 a , b , d , e independent biological replicates with similar results per condition
    Pcr Clean Up Kit, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 92 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcr clean up kit/product/TaKaRa
    Average 99 stars, based on 92 article reviews
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    99
    TaKaRa pcr clean up
    Rat floxed alleles generated by zygote electroporation. a Schematic representation of CLICK: CRISPR with lssDNA inducing conditional knockout alleles. b Schematic approach to generate rat Vapb floxed alleles using the CRISPR/Cas system with lssDNA composed of the targeted exon flanked by two loxP sites (Additional file 11 : Figure S11). c <t>PCR</t> analysis of rat pups (#1–6) generated by CLICK, showing different types of mutations, indels, LD (via NHEJ), inversions, and floxed alleles (via HDR) (black arrow). The primer sets were used for PCR and sequence analysis (Additional file 2 : Figure S2). M: 100 bp <t>DNA</t> ladder marker. d Sequence analysis of 6 pups showing a variety of mutations, indels, inversion or LD indicated by red letters, and loxP insertions and flox indicated by orange letters. All pups were used for testing germline transmission (Additional file 15 : Table S1). Asterisks indicate pups used for testing the Cre-loxP system (Additional file 17 : Table S3)
    Pcr Clean Up, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 38 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcr clean up/product/TaKaRa
    Average 99 stars, based on 38 article reviews
    Price from $9.99 to $1999.99
    pcr clean up - by Bioz Stars, 2020-09
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    Essential role of PRAMEF12 in spermatogonial differentiation. a Immunofluorescence of whole-mount testes from P2, P4, and P7 Pramef12 Null and Pramef12 Het mice after staining with antibodies to DDX4 (left) or KIT (middle) and merged (right). KIT-positive spermatogonia were rarely detected at P2 in either Pramef12 Null or Pramef12 Het tubules but were present at P4 and increased at P7 in Pramef12 Het tubules. Few KIT-positive spermatogonia (arrow) were observed in P7 Pramef12 Null tubules. Scale bar, 50 μm. b Immunohistochemistry of sections from P7 Pramef12 Null (top panels) and Pramef12 Het (bottom panels) testes after staining with antibodies to DDX4 (left) or KIT (middle) and merged with Hoechst 33342 to stain DNA (right). Arrowheads, KIT-positive cells in Pramef12 Null testes. Scale bar, 50 μm. c Quantitative real-time RT-PCR of Kit mRNA abundance in P7 Pramef12 Null and Pramef12 Het testes using β-actin as an internal load control and setting the abundance in Pramef12 Het testis to 1. Mean ± s.d, n = 3 biologically independent samples per condition. * P = 1.43E-05 by two-tailed Student’s t test. d Immunoblot of KIT protein in P7 Pramef12 Null and Pramef12 Het testes using α-tubulin as a load control. e Same as a , but with antibodies to SOHLH1, SOHLH2, and PLZF. The number of spermatogonia positive for SOHLH1 and SOHLH2 was significantly reduced in P7 Pramef12 Null tubules (top panels). Representative of n = 3 a , b , d , e independent biological replicates with similar results per condition

    Journal: Nature Communications

    Article Title: Sertoli cell-only phenotype and scRNA-seq define PRAMEF12 as a factor essential for spermatogenesis in mice

    doi: 10.1038/s41467-019-13193-3

    Figure Lengend Snippet: Essential role of PRAMEF12 in spermatogonial differentiation. a Immunofluorescence of whole-mount testes from P2, P4, and P7 Pramef12 Null and Pramef12 Het mice after staining with antibodies to DDX4 (left) or KIT (middle) and merged (right). KIT-positive spermatogonia were rarely detected at P2 in either Pramef12 Null or Pramef12 Het tubules but were present at P4 and increased at P7 in Pramef12 Het tubules. Few KIT-positive spermatogonia (arrow) were observed in P7 Pramef12 Null tubules. Scale bar, 50 μm. b Immunohistochemistry of sections from P7 Pramef12 Null (top panels) and Pramef12 Het (bottom panels) testes after staining with antibodies to DDX4 (left) or KIT (middle) and merged with Hoechst 33342 to stain DNA (right). Arrowheads, KIT-positive cells in Pramef12 Null testes. Scale bar, 50 μm. c Quantitative real-time RT-PCR of Kit mRNA abundance in P7 Pramef12 Null and Pramef12 Het testes using β-actin as an internal load control and setting the abundance in Pramef12 Het testis to 1. Mean ± s.d, n = 3 biologically independent samples per condition. * P = 1.43E-05 by two-tailed Student’s t test. d Immunoblot of KIT protein in P7 Pramef12 Null and Pramef12 Het testes using α-tubulin as a load control. e Same as a , but with antibodies to SOHLH1, SOHLH2, and PLZF. The number of spermatogonia positive for SOHLH1 and SOHLH2 was significantly reduced in P7 Pramef12 Null tubules (top panels). Representative of n = 3 a , b , d , e independent biological replicates with similar results per condition

    Article Snippet: After linearization by digestion with DraI, the DNA fragment was purified with PCR Clean-up Kit (Clontech Laboratories) and transcribed using AmpliScribe T7-Flash Transcription Kit (Lucigen).

    Techniques: Immunofluorescence, Mouse Assay, Staining, Immunohistochemistry, Quantitative RT-PCR, Two Tailed Test

    Rat floxed alleles generated by zygote electroporation. a Schematic representation of CLICK: CRISPR with lssDNA inducing conditional knockout alleles. b Schematic approach to generate rat Vapb floxed alleles using the CRISPR/Cas system with lssDNA composed of the targeted exon flanked by two loxP sites (Additional file 11 : Figure S11). c PCR analysis of rat pups (#1–6) generated by CLICK, showing different types of mutations, indels, LD (via NHEJ), inversions, and floxed alleles (via HDR) (black arrow). The primer sets were used for PCR and sequence analysis (Additional file 2 : Figure S2). M: 100 bp DNA ladder marker. d Sequence analysis of 6 pups showing a variety of mutations, indels, inversion or LD indicated by red letters, and loxP insertions and flox indicated by orange letters. All pups were used for testing germline transmission (Additional file 15 : Table S1). Asterisks indicate pups used for testing the Cre-loxP system (Additional file 17 : Table S3)

    Journal: BMC Genomics

    Article Title: CLICK: one-step generation of conditional knockout mice

    doi: 10.1186/s12864-018-4713-y

    Figure Lengend Snippet: Rat floxed alleles generated by zygote electroporation. a Schematic representation of CLICK: CRISPR with lssDNA inducing conditional knockout alleles. b Schematic approach to generate rat Vapb floxed alleles using the CRISPR/Cas system with lssDNA composed of the targeted exon flanked by two loxP sites (Additional file 11 : Figure S11). c PCR analysis of rat pups (#1–6) generated by CLICK, showing different types of mutations, indels, LD (via NHEJ), inversions, and floxed alleles (via HDR) (black arrow). The primer sets were used for PCR and sequence analysis (Additional file 2 : Figure S2). M: 100 bp DNA ladder marker. d Sequence analysis of 6 pups showing a variety of mutations, indels, inversion or LD indicated by red letters, and loxP insertions and flox indicated by orange letters. All pups were used for testing germline transmission (Additional file 15 : Table S1). Asterisks indicate pups used for testing the Cre-loxP system (Additional file 17 : Table S3)

    Article Snippet: Bands corresponding to a single-strand DNA fragment were extracted using NucleoSpin® Gel and PCR Clean-up (Takara Bio, Shiga, Japan).

    Techniques: Generated, Electroporation, CRISPR, Knock-Out, Polymerase Chain Reaction, Non-Homologous End Joining, Sequencing, Marker, Transmission Assay

    One-step generation of conditional knockout animals (F0) by CLICK. a Schematic representation of applying CLICK in oocytes for in vitro fertilization (IVF) with Cre-driver mice, Emx1-cre, resulting in brain-specific recombination at the targeted floxed alleles. b PCR analysis of representative delivered mouse pups by microinjection (#1–8) or electroporation (#9–15) showing different types of mutations, indels, LD, and floxed alleles (black arrow) at the targeted Serpina3n locus. The primer sets (‘small’ in Additional file 2 : Figure S2) were used for PCR and sequence analysis. c Genotyping in several tissues: cerebrum (Cr), cerebellum (Cl), heart (H), liver (L), spleen (S), and testis (Ts), indicating recombination (red arrow) by brain-specific Cre expression in #14 mouse carrying heterozygous floxed with LD alleles. M: 100 bp DNA ladder marker. d Time-schedule comparisons of targeting methods using ES cells by CRISPR in B6 oocytes, and CLICK in Cre oocytes. CLICK saves about 6 months of crossing and reduces breeding costs during the study period (Additional file 13 : Figure S13)

    Journal: BMC Genomics

    Article Title: CLICK: one-step generation of conditional knockout mice

    doi: 10.1186/s12864-018-4713-y

    Figure Lengend Snippet: One-step generation of conditional knockout animals (F0) by CLICK. a Schematic representation of applying CLICK in oocytes for in vitro fertilization (IVF) with Cre-driver mice, Emx1-cre, resulting in brain-specific recombination at the targeted floxed alleles. b PCR analysis of representative delivered mouse pups by microinjection (#1–8) or electroporation (#9–15) showing different types of mutations, indels, LD, and floxed alleles (black arrow) at the targeted Serpina3n locus. The primer sets (‘small’ in Additional file 2 : Figure S2) were used for PCR and sequence analysis. c Genotyping in several tissues: cerebrum (Cr), cerebellum (Cl), heart (H), liver (L), spleen (S), and testis (Ts), indicating recombination (red arrow) by brain-specific Cre expression in #14 mouse carrying heterozygous floxed with LD alleles. M: 100 bp DNA ladder marker. d Time-schedule comparisons of targeting methods using ES cells by CRISPR in B6 oocytes, and CLICK in Cre oocytes. CLICK saves about 6 months of crossing and reduces breeding costs during the study period (Additional file 13 : Figure S13)

    Article Snippet: Bands corresponding to a single-strand DNA fragment were extracted using NucleoSpin® Gel and PCR Clean-up (Takara Bio, Shiga, Japan).

    Techniques: Knock-Out, In Vitro, Mouse Assay, Polymerase Chain Reaction, Electroporation, Sequencing, Expressing, Marker, CRISPR

    Mouse floxed alleles generated by microinjection of two gRNA, Cas9 mRNA and lssDNA into zygotes. a An approach to generate Serpina3n floxed alleles using the CRISPR/Cas system with long single-stranded DNA (lssDNA) composed of the targeted exon flanked by two loxP sites (Additional file 2 : Figure S2). b PCR analysis of representative delivered mouse pups (#1–15) showing different types of mutations, indels, LD, and floxed alleles (black arrow) at the targeted Serpina3n locus. The primer sets (‘small’ in Additional file 2 : Figure S2) were used for PCR and sequence analysis. Asterisks indicate pups used for testing germline transmission (Additional file 15 : Table S1). M: 100 bp DNA ladder marker. c Representative examples of the targeted Serpina3n loci generated by the microinjection of two gRNA (gRNA-1 and gRNA-2), Cas9 mRNA and lssDNA into B6 mouse zygotes (Additional file 3 : Figure S3)

    Journal: BMC Genomics

    Article Title: CLICK: one-step generation of conditional knockout mice

    doi: 10.1186/s12864-018-4713-y

    Figure Lengend Snippet: Mouse floxed alleles generated by microinjection of two gRNA, Cas9 mRNA and lssDNA into zygotes. a An approach to generate Serpina3n floxed alleles using the CRISPR/Cas system with long single-stranded DNA (lssDNA) composed of the targeted exon flanked by two loxP sites (Additional file 2 : Figure S2). b PCR analysis of representative delivered mouse pups (#1–15) showing different types of mutations, indels, LD, and floxed alleles (black arrow) at the targeted Serpina3n locus. The primer sets (‘small’ in Additional file 2 : Figure S2) were used for PCR and sequence analysis. Asterisks indicate pups used for testing germline transmission (Additional file 15 : Table S1). M: 100 bp DNA ladder marker. c Representative examples of the targeted Serpina3n loci generated by the microinjection of two gRNA (gRNA-1 and gRNA-2), Cas9 mRNA and lssDNA into B6 mouse zygotes (Additional file 3 : Figure S3)

    Article Snippet: Bands corresponding to a single-strand DNA fragment were extracted using NucleoSpin® Gel and PCR Clean-up (Takara Bio, Shiga, Japan).

    Techniques: Generated, CRISPR, Polymerase Chain Reaction, Sequencing, Transmission Assay, Marker

    Schematic of iv TRT and ssDNA preparation steps. A dsDNA template can be a PCR product, or a plasmid with a suitable restriction enzyme (RE) site distal to the insertion cassette (Steps 1–2) . The gel on the right shows a plasmid digested with a RE. RNA is synthesized using in vitro transcription (Steps 3–18) . The gel on the right shows an RNA of ~ 900 bases long. cDNA is synthesized by reverse transcription using a reverse primer (Steps 19–23) . The gel on the right shows a sample ssDNA (cDNA). Note that the cDNA preparation typically runs like a smear with a prominent band within the smear. Purification of ssDNA (Steps 24–35) . The image on the left shows the gel after excision of the prominent band (for purification) and the gel on the right shows the purified ssDNA. The GeneRuler DNA Ladder Mix (ThermoFisher Scientific, cat. no. SM0331) was used in all the gels as a DNA size marker.

    Journal: Nature protocols

    Article Title: Easi-CRISPR protocol for creating knock-in and conditional knockout mouse models using long ssDNA donors

    doi: 10.1038/nprot.2017.153

    Figure Lengend Snippet: Schematic of iv TRT and ssDNA preparation steps. A dsDNA template can be a PCR product, or a plasmid with a suitable restriction enzyme (RE) site distal to the insertion cassette (Steps 1–2) . The gel on the right shows a plasmid digested with a RE. RNA is synthesized using in vitro transcription (Steps 3–18) . The gel on the right shows an RNA of ~ 900 bases long. cDNA is synthesized by reverse transcription using a reverse primer (Steps 19–23) . The gel on the right shows a sample ssDNA (cDNA). Note that the cDNA preparation typically runs like a smear with a prominent band within the smear. Purification of ssDNA (Steps 24–35) . The image on the left shows the gel after excision of the prominent band (for purification) and the gel on the right shows the purified ssDNA. The GeneRuler DNA Ladder Mix (ThermoFisher Scientific, cat. no. SM0331) was used in all the gels as a DNA size marker.

    Article Snippet: 28) Extract DNA from the gel slice using NucleoSpin Gel or PCR Clean-up (TaKaRa) kit (option A) or by Phenol extraction and ethanol precipitation (option B).

    Techniques: Polymerase Chain Reaction, Plasmid Preparation, Synthesized, In Vitro, Purification, Marker