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    Thermo Fisher pcr kit
    Generation of a mouse model for conditional PAK4 gene depletion in the pancreas. ( a ) Graphical representation of our strategy for generation of a mouse model with conditional PAK4 gene depletion in the pancreas. Exons are indicated by light grey rectangles and Lox P sites are indicated by black triangles. Pdx1-driven Cre expression and consequent recombination of LoxP sites results in the depletion of exons 2–4 in the mouse PAK4 gene as previously described 26 . ( b ) <t>PCR</t> analysis of genomic <t>DNA</t> from 12 days old mouse tails. Upper panel shows the presence of the Pdx1-Cre allele and the lower panel shows PAK4; the upper band in the lower panel displays the floxed PAK4 allele, while the lower band in the lower panel displays a PAK4 WT allele. Thus, appearance of the upper band alone displays homozygous floxed PAK4 allele; the lower band alone represents WT PAK4 allele; while both bands together mean that the mouse is PAK4 heterozygous, i.e. one WT and one floxed allele. Full gels are showed in supplementary Fig. S1a and S1b . ( c ) PAK4 deletion in the mouse pancreas. Immunoblot analysis of pancreatic whole cell lysates shows PAK4 expression in WT pancreas, whereas PAK4 protein expression is below the detection limit in PAK4 homozygous KO mice. Vinculin was used as a loading control. *Shows unspecific bands. Full blot is showed in supplementary Fig. S1c .
    Pcr Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2594 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Generation of a mouse model for conditional PAK4 gene depletion in the pancreas. ( a ) Graphical representation of our strategy for generation of a mouse model with conditional PAK4 gene depletion in the pancreas. Exons are indicated by light grey rectangles and Lox P sites are indicated by black triangles. Pdx1-driven Cre expression and consequent recombination of LoxP sites results in the depletion of exons 2–4 in the mouse PAK4 gene as previously described 26 . ( b ) PCR analysis of genomic DNA from 12 days old mouse tails. Upper panel shows the presence of the Pdx1-Cre allele and the lower panel shows PAK4; the upper band in the lower panel displays the floxed PAK4 allele, while the lower band in the lower panel displays a PAK4 WT allele. Thus, appearance of the upper band alone displays homozygous floxed PAK4 allele; the lower band alone represents WT PAK4 allele; while both bands together mean that the mouse is PAK4 heterozygous, i.e. one WT and one floxed allele. Full gels are showed in supplementary Fig. S1a and S1b . ( c ) PAK4 deletion in the mouse pancreas. Immunoblot analysis of pancreatic whole cell lysates shows PAK4 expression in WT pancreas, whereas PAK4 protein expression is below the detection limit in PAK4 homozygous KO mice. Vinculin was used as a loading control. *Shows unspecific bands. Full blot is showed in supplementary Fig. S1c .

    Journal: Scientific Reports

    Article Title: Pdx1-Cre-driven conditional gene depletion suggests PAK4 as dispensable for mouse pancreas development

    doi: 10.1038/s41598-017-07322-5

    Figure Lengend Snippet: Generation of a mouse model for conditional PAK4 gene depletion in the pancreas. ( a ) Graphical representation of our strategy for generation of a mouse model with conditional PAK4 gene depletion in the pancreas. Exons are indicated by light grey rectangles and Lox P sites are indicated by black triangles. Pdx1-driven Cre expression and consequent recombination of LoxP sites results in the depletion of exons 2–4 in the mouse PAK4 gene as previously described 26 . ( b ) PCR analysis of genomic DNA from 12 days old mouse tails. Upper panel shows the presence of the Pdx1-Cre allele and the lower panel shows PAK4; the upper band in the lower panel displays the floxed PAK4 allele, while the lower band in the lower panel displays a PAK4 WT allele. Thus, appearance of the upper band alone displays homozygous floxed PAK4 allele; the lower band alone represents WT PAK4 allele; while both bands together mean that the mouse is PAK4 heterozygous, i.e. one WT and one floxed allele. Full gels are showed in supplementary Fig. S1a and S1b . ( c ) PAK4 deletion in the mouse pancreas. Immunoblot analysis of pancreatic whole cell lysates shows PAK4 expression in WT pancreas, whereas PAK4 protein expression is below the detection limit in PAK4 homozygous KO mice. Vinculin was used as a loading control. *Shows unspecific bands. Full blot is showed in supplementary Fig. S1c .

    Article Snippet: Genotyping by PCR Genomic DNA was extracted from 12–19 day old mouse tails using the fast tissue–to-PCR kit (#K1091, Fermentas).

    Techniques: Expressing, Polymerase Chain Reaction, Mouse Assay

    Establishment of HCKT ‐E7‐ HRAS G 12V expressing the wild type HPV 16E6 or its mutants defective for degradation of p53 and/or PDZ domain containing proteins. Expression of HPV 16E7, HRAS mutant, HRAS G 12V , and HPV 16E6 or its mutants was introduced to HCK 1T cells by retrovirus mediated transduction as described in Materials and Method. The levels of the wild type 16E6 and its mutant were examined by Western blots (a) and RT ‐ PCR (b). (a) The mouse monoclonal antibody for HPV 16E6 was raised against 16 amino acids of its N‐terminal region and therefore does not react with HPV 16E6 SAT which contains R8S/P9A/R10T substitution. While the wild type E6 and E6Δ151 which lacks its C‐terminal amino acid induced p53 degradation, E6 mutants with SAT substituions such as E6 SAT and E6 SAT Δ151 did not do so. The levels of PDZ domain containing proteins, such as Scribble ( SCRIB ), DLG 4, MAGI ‐1 ( MAGI ) and PAR 3 were decreased in wild‐type E6, but not E6Δ151 or E6 SAT Δ151 expressing cells. The level of HPV 16E7 and HRAS were not affected by the expression of E6. α‐tubulin was detected as a loading control. (b) mRNA levels of the wild type E6 and its mutants were comparable in those cells. 36B4 mRNA was also detected as an internal control. (c) The level of PTPN 13 was compared in indicated cells by Western blotting.

    Journal: Cancer Science

    Article Title: Roles of the PDZ‐binding motif of HPV 16 E6 protein in oncogenic transformation of human cervical keratinocytes

    doi: 10.1111/cas.13264

    Figure Lengend Snippet: Establishment of HCKT ‐E7‐ HRAS G 12V expressing the wild type HPV 16E6 or its mutants defective for degradation of p53 and/or PDZ domain containing proteins. Expression of HPV 16E7, HRAS mutant, HRAS G 12V , and HPV 16E6 or its mutants was introduced to HCK 1T cells by retrovirus mediated transduction as described in Materials and Method. The levels of the wild type 16E6 and its mutant were examined by Western blots (a) and RT ‐ PCR (b). (a) The mouse monoclonal antibody for HPV 16E6 was raised against 16 amino acids of its N‐terminal region and therefore does not react with HPV 16E6 SAT which contains R8S/P9A/R10T substitution. While the wild type E6 and E6Δ151 which lacks its C‐terminal amino acid induced p53 degradation, E6 mutants with SAT substituions such as E6 SAT and E6 SAT Δ151 did not do so. The levels of PDZ domain containing proteins, such as Scribble ( SCRIB ), DLG 4, MAGI ‐1 ( MAGI ) and PAR 3 were decreased in wild‐type E6, but not E6Δ151 or E6 SAT Δ151 expressing cells. The level of HPV 16E7 and HRAS were not affected by the expression of E6. α‐tubulin was detected as a loading control. (b) mRNA levels of the wild type E6 and its mutants were comparable in those cells. 36B4 mRNA was also detected as an internal control. (c) The level of PTPN 13 was compared in indicated cells by Western blotting.

    Article Snippet: Fifty nanograms of cDNA template was subjected to PCR amplification with primer sets specific to E6 or acidic ribosomal phosphoprotein P0 (36B4) using a PCR reagents kit (Applied Biosystems, Foster City, CA, USA).

    Techniques: Expressing, Mutagenesis, Transduction, Western Blot, Reverse Transcription Polymerase Chain Reaction

    FOXP3 mRNA relative expression level in lung tissue in each group at 4, 8 and 24h. Six groups were studied: Control group, IS group, PA group, IS + PA group, PA + IVIG group and IS + PA + IVIG group. Immunosuppression was induced on day -5, -3 and -1 by intraperitoneal injection of CYP, PA pneumonia was induced on day 0 and IVIG mediated treatment models were immediately established following PA intervention on day 0. The left lung tissue of the mice was used for testing FOXP3 mRNA relative expression by qRT-PCR. qRT-PCR was carried out using a Thermo Fisher PCR kit with FOXP3 and GAPDH primer. FOXP3 mRNA relative expression level was calculated by the 2 (- ΔΔ CT) method. FOXP3 mRNA expression levels in lung tissue in six groups at 4, 8 and 24h were shown.

    Journal: PLoS ONE

    Article Title: Effect of intravenous immunoglobulin on the function of Treg cells derived from immunosuppressed mice with Pseudomonas aeruginosa pneumonia

    doi: 10.1371/journal.pone.0176843

    Figure Lengend Snippet: FOXP3 mRNA relative expression level in lung tissue in each group at 4, 8 and 24h. Six groups were studied: Control group, IS group, PA group, IS + PA group, PA + IVIG group and IS + PA + IVIG group. Immunosuppression was induced on day -5, -3 and -1 by intraperitoneal injection of CYP, PA pneumonia was induced on day 0 and IVIG mediated treatment models were immediately established following PA intervention on day 0. The left lung tissue of the mice was used for testing FOXP3 mRNA relative expression by qRT-PCR. qRT-PCR was carried out using a Thermo Fisher PCR kit with FOXP3 and GAPDH primer. FOXP3 mRNA relative expression level was calculated by the 2 (- ΔΔ CT) method. FOXP3 mRNA expression levels in lung tissue in six groups at 4, 8 and 24h were shown.

    Article Snippet: Total cDNA was produced by reverse transcription via a Thermo Fisher reverse transcription kit (purchased from Thermo Fisher Scientific). qRT-PCR was carried out using a Thermo Fisher PCR kit with FOXP3 and glyceradehyde-3-phosphate-dehydrogenase (GAPDH) primers (synthesized by TaKaRa Biological Engineering Co., Ltd. Shanghai, China).

    Techniques: Expressing, Injection, Mouse Assay, Quantitative RT-PCR, Polymerase Chain Reaction