End-point PCR amplification was performed using JumpStart RedTaq (Sigma). The Applied Biosystems Veriti 96-well thermal cycler (Applied Biosystems) and Gel DOC EZ imager (Bio-Rad) were used analyses. Gene expression was quantified by real-time qPCR in CFX96 Normal-Well Real-Time System (BioRad) using PerfeCta SYBR Green FastMix and specific oligonucleotide primers. The reaction mixtures contained 10 μl PerfeCta SYBR Green FastMix, 7.2 μl ddH2O, 2.0 μl template cDNA and 0.4 μl gene-specific 10 μM PCR oligonucleotides primers. The reaction conditions were 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 60°C for 30 s and Melt Curve (dissociation stage). Relative gene expression was calculated as delta (Δ Re (the difference between the cycle threshold values, Ct, of the internal control, and Ct of gene of interest) and confirmed by 2–ΔΔ CT method.
Jumpstart redtaq
Manufactured by Merck Group
Sourced in United States
JumpStart RedTaq is a polymerase enzyme used in polymerase chain reaction (PCR) experiments. It is a thermostable DNA polymerase that enables rapid amplification of DNA fragments. The product is designed to provide reliable and efficient DNA amplification for a wide range of applications.
Automatically generated - may contain errors
Lab products found in correlation
Gel doc ez imager, by Bio-Rad (4 mentions)
Veriti 96 well thermal cycler, by Thermo Fisher Scientific (3 mentions)
Gentra puregene tissue kit, by Qiagen (2 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (2 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
Perfecta sybr green fastmix, by Bio-Rad (1 mentions)
C57bl 6j mice, by Jackson ImmunoResearch (1 mentions)
Pgem t easy vector system 2, by Promega (1 mentions)
Epitect bisulfite kit, by Qiagen (1 mentions)
Qiaprep spin miniprep kit, by Qiagen (1 mentions)
Cfx 96 well real time pcr system, by Bio-Rad (1 mentions)
Perfecta sybr green fastmix rox, by Quanta Biosciences (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Veriti thermocycler, by Thermo Fisher Scientific (1 mentions)
M mlv reverse transcriptase, by Promega (1 mentions)
Geldoc uv transilluminator, by Bio-Rad (1 mentions)
Geneamp 2700 thermal cycler, by Thermo Fisher Scientific (1 mentions)
Cfx96 deep well real time system, by Bio-Rad (1 mentions)
Molf eij, by Jackson ImmunoResearch (1 mentions)
C57bl 6j, by Jackson ImmunoResearch (1 mentions)
15 protocols using jumpstart redtaq
1
Quantitative gene expression analysis by real-time qPCR
2
Mouse Genomic DNA Extraction and Genotyping
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Genomic DNA was prepared from mouse tail tissue using the Gentra Puregene Tissue Kit (Qiagen). PCR amplification was performed using Jumpstart RedTaq (Sigma-Aldrich). Primer sets (Table S1 ) are as follows: For all mice: neo (Neo-F/R) and Crx (Total Crx-F/R); for E168d2 colony: WT Crx allele (E168d2 WT-F, E168d2-R), E168d2 allele (E168d2 Mut-F, E168d2-R); for R90W colony: WT Crx allele (R90W WT-R, R90W-R), R90W allele (R90W Mut-F, R90W-R).
3
Generating Genetically Modified Mouse Models
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All mice used in this study were backcrossed to C57BL/6 J mice (the Jackson Laboratory, Bar Harbor, Maine; Stock # 000664) and housed in Washington University School of Medicine barrier facilities. Mll1 flox mice29 (link) were obtained from Patricia Ernst at University of Colorado, Denver. Chx10-Cre37 (link) mice were provided by Connie Cepko at Harvard University. Both females and males were included in each experiment.
For genotyping, genomic DNA was isolated from mouse tail clips using the Gentra Puregene Tissue Kit (Qiagen, Germantown, MD). PCR amplification was performed using Jumpstart RedTaq (Sigma-Aldrich, St. Louis, MO) and PCR primers as published for each mouse.
All animal procedures were conducted according to the Guide for the Care and Use of Laboratory Animals of the National Institute of Health, and were approved by the Washington University in St. Louis Institutional Animal Care and Use Committee.
For genotyping, genomic DNA was isolated from mouse tail clips using the Gentra Puregene Tissue Kit (Qiagen, Germantown, MD). PCR amplification was performed using Jumpstart RedTaq (Sigma-Aldrich, St. Louis, MO) and PCR primers as published for each mouse.
All animal procedures were conducted according to the Guide for the Care and Use of Laboratory Animals of the National Institute of Health, and were approved by the Washington University in St. Louis Institutional Animal Care and Use Committee.
4
Bisulfite Sequencing of 4qA-L Allele
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DNA methylation was analyzed by bisulfite conversion (EpiTect Bisulfite Kit, Qiagen) of genomic DNA from differentiated myocytes. To perform the bisulfite analysis of the 4qA-L allele, previously published [21 (link)] PCR oligonucleotide primers (BSS4qALF for forward 5′-TTATTTATGAAGGGGTGGAGTTTGTT and BSS3626R for reverse 5′-AACAAAAATATACTTTTAACCRCCAAAAA) were utilized. All PCRs were performed using the hotstart Taq (JumpStart REDTaq from Sigma) at 95 °C for 5 min, 95 °C for 30 s, 55 °C for 30 s, 40 cycles at 72 °C for 1 min and 72 °C for 1 min. The amplified 354-bp PCR product was then gel purified and cloned into pGEM-T-Easy vector system II (Promega) and transformed into DH10B electrocompetent cells. The DH10B cells that carried the ligated vectors were selected by color change on agar plates containing the ampicillin/X-gal/IPTG. The white colonies represented the vectors with the PCR product, while the blue colonies represented empty vectors. The white colonies were picked and bulked in LB medium. The plasmids containing the target DNA were extracted by using the QIAprep Spin Miniprep Kit (Qiagen) and sequenced to reveal methylation distribution at a single molecule level.
5
Quantifying Human Frizzled Receptors
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For endpoint PCR analysis intron-spanning primers specific for human frizzled receptor 1–9 and β-actin were used (Table 1 ). Total RNA was isolated using Trizol (Invitrogen), and 2 µg of RNA was reverse-transcribed with M-MLV Reverse Transcriptase (Promega). Endpoint PCR reactions utilized JumpStart RedTaq (Sigma) on Veriti thermocycler (Applied biosystems). Real-time PCR Reactions for were ran on CFX96 well Real-Time PCR System (Bio-Rad) and utilized PerfeCTa SYBR green FastMix, ROX (Quanta Bioscience, Inc., MD, cat. number 95073-012).
6
Multiplex PCR for Listeria Identification
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A multiplex PCR was developed with the aim to identify the six species of Listeria (L. monocytogenes, L. innocua, L. ivanovii, L. grayi, L. seeligeri and L. welshimeri) and Listeria spp., by using the PCR protocol described by Ryu et al. (2013 ) that has been partially modified in order to obtain the seven differentials bands for each Listeria species in a single reaction. Primers concentrations are reported in Table 1 . All amplification reactions were performed in a final volume of 25 µL containing 5 µL of DNA, 5 µL of 10X PCR buffer (JumpStart REDTq DNA Polymerase, Sigma-Aldrich, St. Louis, MO, USA), 4 mM of MgCl2, 0.1 mM each of dNTP, and 2 U of JumpStart RED Taq (Sigma-Aldrich). All amplification reactions were performed in a Gene-Amp 2700 Thermal Cycler (Applied Biosystems, Foster City, CA, USA) programmed as follows: denaturation at 94°C for 5 min, annealing at 58°C for 30 sec and elongation at 72°C for 30 sec, followed by a final extension period at 72°C for 5 min. The amplified fragments were separated by 3% agarose gel electrophoresis (Roche diagnostics, Milan, Italy) in 1X Tris-acetate EDTA (TAE; Invitrogen, Carlsbad, CA, USA) and stained with ethidium bromide (0.1 mg/mL) for 20 min. The gels were observed and the images acquired by the Gel-Doc UV trans-illuminator (Bio-Rad, Hercules, CA, USA).
7
RNA Isolation and Gene Expression Analysis
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Total RNA was isolated from cells using a Pure-link RNA mini kit (Invitrogen). 2 μg of total RNA was reverse-transcribed using SuperScriptIII Reverse Transcriptase (ThermoFisher) to synthesize first-strand of complementary DNA (cDNA). Intron-spanning primers were designed for each specific target DNA and gene expression measured by endpoint-PCR using JumpStart RedTaq (Sigma). The primers listed in Table 1 were used for expression analysis for Fig. 1A and 8C .
Real time qPCR primers used for the study.
| Forward Primer | Reverse primer | |
|---|---|---|
| DVL-1 | 5′-GGGAGTCAGCAGAGTGAAGG-3′ | 5′-CACCACTGTATAGGCCTTGGTC-3′ |
| I.4 promoter | 5′-GAGGTCACAGAAGGCAGAGG-3′ | 5′-GAGGGGGCAATTTAGAGTCC-3′ |
| Total aromatase | 5′-GGGATCGGCAGTGCCTGCAA-3′ | 5′-AACAAGGCCGGGGCCTGACA-3′ |
| Beta actin | 5′-GGACTTCGAGCAAGAGATGG-3′ | 5′-AGCACTGTGTTGGCGTACAG-3′ |
8
End-Point PCR and Real-Time qPCR Protocol
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End-point PCR amplification was performed using JumpStart RedTaq (Sigma). The Applied Biosystems Veriti 96-well thermal cycler (Applied Biosystems) and Gel DOC EZ imager (Bio-Rad) were used analyses. Gene expression was quantified by real-time qPCR in CFX96 Deep-Well Real-Time System (BioRad) using PerfeCta SYBR Green FastMix and specific oligonucleotide primers. The reaction mixtures contained 10 µl PerfeCta SYBR Green FastMix, 7.2 µl ddH2O, 2.0 µl template cDNA and 0.4 µl gene-specific 10 µM PCR oligonucleotides primers. The reaction conditions were 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s and Melt Curve (dissociation stage). Relative gene expression was calculated as delta (∆ Re (the difference between the cycle threshold values, Ct, of the internal control, and Ct of gene of interest) and confirmed by 2–∆∆ CT method.
9
Genetic Mapping of STING Pathway
10
RNA Extraction and RT-qPCR Analysis
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