expand high fidelity pcr system (Roche)


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![Replacement of SENP. ( A ) Schematic representation of LdBPK_262070 replacement using the <t>CRISPR/Cas9</t> technology. SUMO -targeting sgRNAs (grey arrowheads) and replacement cassettes were <t>PCR-amplified</t> and transfected into L. donovani (Cas9/T7RNAP). ( B , C ) RT-qPCR of RNA from L. donovani wild type (WT), SENP −/− clones 1–4, SENP −/− cl.1[pCLN-SENP], and L. donovani (Cas9/T7RNAP). ( B ) SENP-specific RT-qPCR. ( C ) Cas9-specific RT-qPCR. n = 2. ( D ) Whole genome sequencing of L. donovani wild type (WT), L. donovani (Cas9/T7RNAP), SENP −/− cl.1 and SENP −/− cl.2. Sequence reads were aligned to L. donovani chromosome 26. The ruler shows the position of the SENP CDS; the numbers refer to the position within chromosome 26. Read coverage is shown in blue.](https://storage.googleapis.com/bioz_article_images/PMC7602377/genes-11-01198-g003.jpg)
Expand High Fidelity Pcr System, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 1479 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "The Leishmania donovani SENP Protease Is Required for SUMO Processing but Not for Viability"
Article Title: The Leishmania donovani SENP Protease Is Required for SUMO Processing but Not for Viability
Journal: Genes
doi: 10.3390/genes11101198
![... -targeting sgRNAs (grey arrowheads) and replacement cassettes were PCR-amplified and transfected into L. donovani (Cas9/T7RNAP). ( B ... Replacement of SENP. ( A ) Schematic representation of LdBPK_262070 replacement using the CRISPR/Cas9 technology. SUMO -targeting sgRNAs (grey arrowheads) and replacement cassettes were PCR-amplified and transfected into L. donovani (Cas9/T7RNAP). ( B , C ) RT-qPCR of RNA from L. donovani wild type (WT), SENP −/− clones 1–4, SENP −/− cl.1[pCLN-SENP], and L. donovani (Cas9/T7RNAP). ( B ) SENP-specific RT-qPCR. ( C ) Cas9-specific RT-qPCR. n = 2. ( D ) Whole genome sequencing of L. donovani wild type (WT), L. donovani (Cas9/T7RNAP), SENP −/− cl.1 and SENP −/− cl.2. Sequence reads were aligned to L. donovani chromosome 26. The ruler shows the position of the SENP CDS; the numbers refer to the position within chromosome 26. Read coverage is shown in blue.](https://storage.googleapis.com/bioz_article_images/PMC7602377/genes-11-01198-g003.jpg)
Figure Legend Snippet: Replacement of SENP. ( A ) Schematic representation of LdBPK_262070 replacement using the CRISPR/Cas9 technology. SUMO -targeting sgRNAs (grey arrowheads) and replacement cassettes were PCR-amplified and transfected into L. donovani (Cas9/T7RNAP). ( B , C ) RT-qPCR of RNA from L. donovani wild type (WT), SENP −/− clones 1–4, SENP −/− cl.1[pCLN-SENP], and L. donovani (Cas9/T7RNAP). ( B ) SENP-specific RT-qPCR. ( C ) Cas9-specific RT-qPCR. n = 2. ( D ) Whole genome sequencing of L. donovani wild type (WT), L. donovani (Cas9/T7RNAP), SENP −/− cl.1 and SENP −/− cl.2. Sequence reads were aligned to L. donovani chromosome 26. The ruler shows the position of the SENP CDS; the numbers refer to the position within chromosome 26. Read coverage is shown in blue.
Techniques Used: CRISPR, Polymerase Chain Reaction, Amplification, Transfection, Quantitative RT-PCR, Clone Assay, Sequencing

Figure Legend Snippet: Replacement of SUMO in Leishmania donovani. ( A ) Schematic representation of LdBPK_080480.1 replacement using the CRISPR/Cas9 technology. SUMO -targeting sgRNAs (grey) and the replacement cassettes were PCR-amplified and transfected into a Cas9/T7-RNAP-expressing L. donovani strain. Two sets of genotyping primers were used to test for the presence of the gene of interest (GOI) ( B ) Gene-specific replacement cassettes amplified from pTPURO or pTBLAST vector were analyzed by agarose gel electrophoresis and ethidium bromide staining. The position of the DNA size marker is indicated on the left, the primers used are indicated on the right. ( C ) Amplified sgRNA-coding sequences were separated on a 1% agarose gel and stained with ethidium bromide. ( D ) Genotyping of putative gene replacement mutant clones with primer pairs 7+8 or 5+6. PCR products were analyzed by 1% agarose gel electrophoresis. Positions of DNA size markers are shown to the left; the primer pairs are indicated on the right. ( E ) Genotyping of gene replacement mutants in the SUMO over expression background (SUMO −/−/+ ) indicated primer pairs.
Techniques Used: CRISPR, Polymerase Chain Reaction, Amplification, Transfection, Expressing, Plasmid Preparation, Agarose Gel Electrophoresis, Staining, Marker, Mutagenesis, Clone Assay, Over Expression
2) Product Images from "B-Cell-Specific Myd88 L252P Expression Causes a Premalignant Gammopathy Resembling IgM MGUS"
Article Title: B-Cell-Specific Myd88 L252P Expression Causes a Premalignant Gammopathy Resembling IgM MGUS
Journal: Frontiers in Immunology
doi: 10.3389/fimmu.2020.602868

Figure Legend Snippet: CDR3 analysis of rearranged VDJ genes shows expansion of clonally related plasma cells in aged Cre ERT2 ;Myd88 L252P mice. Genomic DNA was purified from GFP-reporter-positive TACI + CD138 + plasma cells isolated from bone marrow and spleen of CD19-Cre ERT2 ;Myd88 L252P mice 70 weeks after tamoxifen induction. (A) PCR amplification of rearranged J558 family V genes from bone marrow (upper panel) and spleen (lower panel). Bands corresponding to all four JH segments appeared in the controls, while in Cre ERT2 ;Myd88 L252P mice only J H 4 rearrangements could be detected. (No rearrangements were detected in mouse #2). (B) J H 4 bands (red rectangle shown in A ) were cloned and sequenced. Clonal analysis based on CDR3 sequence revealed that in each mouse the same clones were most frequently detected in the bone marrow (upper panel) and spleen (lower panel). For the most frequently detected clonotype (red sector in pie chart), the % of sequences detected and the V H J558 family member is given. V H genes and VDJ rearrangements of the most frequent clonotypes were shared in bone marrow and spleen of each CD19‑Cre ERT2 ;Myd88 L252P mouse, while they differed in bone marrow and spleen of controls and between the controls. Each pie chart represents one mouse. (See also Supplementary Figures 9 and 10 ).
Techniques Used: Mouse Assay, Purification, Isolation, Polymerase Chain Reaction, Amplification, Clone Assay, Sequencing
3) Product Images from "Cu transporter protein CrpF protects against Cu-induced toxicity in Fusarium oxysporum"
Article Title: Cu transporter protein CrpF protects against Cu-induced toxicity in Fusarium oxysporum
Journal: Virulence
doi: 10.1080/21505594.2020.1809324

Figure Legend Snippet: Transcriptional analyses of crpF , metal homeostasis mt1 and aceA and stress prx and gapdh related genes by RT-PCR analysis. Transcript levels of act, mt1, crpF, aceA, prx and gapdh from wt and Δ crpF strains on control condition and under exposure to 0.1 mM CdCl 2, 0.175 mM CuSO 4 or 7.5 mM ZnCl 2 are shown.
Techniques Used: Reverse Transcription Polymerase Chain Reaction

Figure Legend Snippet: Targeted deletion of the F. oxysporum crpF gene. (a) Targeted gene replacement strategy using a disruption construct obtained by fusion PCR with Hyg R cassette as selective marker. Relative positions of primers used for PCR and probe (dashes bars) are indicated. (b) Southern blot analysis of gDNA from wt strain and transformants. gDNAs were digested with Sma I and Sac II to detect deletion and complementation of crpF . The Southern blot image provided comes from two nitrocellulose membranes. The images were manipulated with the objective of only show the interesting transformants.
Techniques Used: Construct, Polymerase Chain Reaction, Marker, Southern Blot
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