Redextract n amp pcr readymix

Manufactured by Merck Group

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REDExtract-N-Amp PCR ReadyMix is a ready-to-use solution for DNA amplification by PCR. It contains all the necessary components, including a DNA polymerase, PCR buffer, and dNTPs, pre-mixed and optimized for efficient and reliable PCR reactions.

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14 protocols using redextract n amp pcr readymix

DNA Extraction and Genotyping of Mouse Tails

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DNA extraction from mouse tail clips obtained at 7–10 days of age was performed using REDExtract-N-Amp™ Tissue PCR Kit (Cat#R4775, Sigma-Aldrich, St. Louis, MO, USA) following the manufacturer’s protocol. In brief, tail clips were combined with 100 μL of extraction solution and 25 μL of tissue preparation solution and mixed by vortexing. Samples were incubated for 15 min at room temperature followed by incubation at 95 °C for 5 min. A total of 100 μL of neutralization solution was added to each sample and again mixed by vortexing.
The extracted DNA was subsequently used to perform genotyping PCR. The primers of interest were combined with the extracted DNA, double-distilled water, and REDExtract-N-Amp™ PCR Ready Mix (Cat#R4775, Sigma-Aldrich, St. Louis, MO, USA) to obtain a total volume of 20 μL per reaction. PCR was performed using an Applied Biosystems Thermal Cycler; individual thermal protocols were primer-specific (Supplementary Table S1). A total of 2.5–8 μL of the final samples were run out on 1.5% agarose gels for BAP1 and Kras specimens and 3% agarose gels for Alb-Cre specimens. Primer sequences and expected amplicon sizes are found in Supplementary Figure S1.

Marcus R., Ferri-Borgogno S., Hosein A., Foo W.C., Ghosh B., Zhao J., Rajapakshe K., Brugarolas J., Maitra A, & Gupta S. (2021). Oncogenic KRAS Requires Complete Loss of BAP1 Function for Development of Murine Intrahepatic Cholangiocarcinoma. Cancers, 13(22), 5709.

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Cre-mediated Recombination for Rb1, p53, Yap, and Taz

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Cre-mediated recombination of floxed Rb1, p53, Yap and Taz alleles was confirmed in retina and lung models by PCR (data not shown, primers and diagnostic fragment sizes are in Table S8). Tissue was scraped from 14 µm-thick OCT-embedded frozen tissue sections on glass slides using a 30 G x 1/2” hypodermic needle and extracted by alkaline lysis (25 mM NaOH, 0.2 mM EDTA, pH 12) with heating at 95^oC for ~1 hr followed by neutralization (40 mM Tris-HCl, pH 5). Rb1 and p53 primers have been described (Meuwissen et al., 2003 (link)), and all primer sequences are listed in Table S8. PCR was performed with 500 nM each primer using REDExtract-N-Amp PCR ReadyMix (Sigma) and the following cycling conditions: 94^oC for 3 min; 35 cycles of 94^oC for 0:40 min, 60^oC (Rb1, p53, Yap and Taz F1-R1) or 63^oC (Taz F2-R2) for 0:40 min, 72^oC for 1 min; 72^oC for 10 min. PCR products were resolved on 2% agarose gels.

Pearson J.D., Huang K., Pacal M., McCurdy S.R., Lu S., Aubry A., Yu T., Wadosky K.M., Zhang L., Wang T., Gregorieff A., Ahmad M., Dimaras H., Langille E., Cole S.P., Monnier P.P., Lok B.H., Tsao M.S., Akeno N., Schramek D., Wikenheiser-Brokamp K.A., Knudsen E.S., Witkiewicz A.K., Wrana J.L., Goodrich D.W, & Bremner R. (2021). Binary pan-cancer classes with distinct vulnerabilities defined by pro- or anti-cancer YAP/TEAD activity. Cancer cell, 39(8), 1115-1134.e12.

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Genetic Characterization of Mouse Models

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Breeding was performed in the IGR or CINJ animal facilities. Tyr::CreERT2 and Pten^fl/fl mice were purchased from JAX (JAX012328 and JAX004597, respectively). BRaf^CA/CA mice were obtained from McMahon's laboratory.⁴⁹ (link)
Atg7^fl/fl mice were provided by Kamatsu's laboratory.⁵⁰ (link)
By the age of 3 weeks, pup tails were partially cut for genotyping. DNA was extracted from using the DNAeasy Blood and Tissue Kit (Quiagen). DNA was then amplified by PCR in Micro-Amp 96-well reaction plate (Applied Biosystems), using REDExtract-N-Amp PCR ready Mix (Sigma). The primers used were 5′-GCG GTC TGG CAG TAA AAA CTA TC-3′, 5′-GTG AAA CAG CT TGC TGT CAC TT-3′, 5′-CTA GGC CAC AGA ATT GAA AGA TCT-3′ and 5′-GTA GGT GGA AAT TCT AGC ATC ATC C-3′ for Tyr-CRE, 5′-AAG CAC TCT GCG AAC TGA G-3′ and 5′-AAG TTT TTG AAG GCA AGA TGC-3′ for PTEN, 5′-TGA GTA TTT TTG TGG CAA CTG C-3′ and 5′-CTC TGC TGG GAA AGC GGC-3′ for BRAF, 5′-TGG CTG CTA CTT CTG CAA TGA TGT-3′ and 5′- CAG GAC AGA GAC CT CAG CTC CAC-3′ for ATG7 (JAX database).^49,50 (link) PCR products were finally loaded on agarose gels and revealed, as previously described (JAX database).

Michaud M., Xie X., Bravo-San Pedro J.M., Zitvogel L., White E, & Kroemer G. (2014). An autophagy-dependent anticancer immune response determines the efficacy of melanoma chemotherapy. Oncoimmunology, 3(7), e944047.

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PCR Detection of mecA Gene in Coagulase-Positive Staphylococci

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The presence of the mecA gene was investigated in all the isolated coagulase-positive oxacillin-resistant staphylococci using PCR according to the modified protocol of Stegger et al. [25 (link)]. Each PCR contained 25 μm of the mecA primer (mecA-P1-5′-TCCAGATTACAACTTCACCAGG-3′; and mecA-P2-5′-CCACTTCATATCTTGTAACG-3′) (162pb), 1x REDExtract-N-Amp PCR ReadyMix (Sigma-Aldrich, Milan, Italy) and 2 μL of DNA template preparation. DNA of Staphylococcus aureus ATCC 48866 and water were used as positive and negative controls, respectively.

Cocca G., Piva S., Magno S.D., Scarpellini R., Giacometti F., Serraino A, & Giunti M. (2021). Prevalence and Patterns of Antimicrobial Resistance among Escherichia coli and Staphylococcus spp. in a Veterinary University Hospital. Veterinary Sciences, 8(12), 308.

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Generation and Genotyping of Zebrafish ak2 Mutants

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The generation of zebrafish ak2 knockout mutants using CompoZr ZFNs (Sigma-Aldrich) and the genotyping strategy have been previously described (Sood et al., 2013 (link)). We selected two mutations predicted to cause frameshifts with premature terminations, ak2^hg14 (c.45delAA, p.K16RfsX62) and ak2^hg15 (c.41insACGG, p.K16TfsX64), denoted here as ak2^del2 and ak2^ins4, respectively. We also recovered a mutant line (ak2^hg16, denoted as ak2^L124P) carrying a c.T371C/p.L124P missense mutation within ak2 exon 4 from a zebrafish DNA library of ENU-induced mutations. DNA extraction and amplification from fixed samples were performed using Extract-N-Amp Tissue PCR kit (Sigma-Aldrich) under standard conditions and REDExtract-N-Amp PCR Ready Mix (Sigma-Aldrich). Genomic sequences flanking the point mutation of ak2^L124P mutants were PCR amplified using the following primers: AK2-F3seq (5′-TGTAAAACGACGGCCAGTTCTCATTTGTAGCTGGATGAC-3′) and AK2-R4seq (5′-CAGGAAACAGCTATGACCCACTTACAGGACCCTCCATGC-3′). PCR products were sequenced with M13R primer and big-dye v3.1 sequencing mix (Life Technologies) after removal of unused primers and nucleotides with Exo-SAP-IT (Affymetrix). Sequence analysis was performed using software package Sequencher, version 5.0 (Gene Codes).

Rissone A., Weinacht K.G., la Marca G., Bishop K., Giocaliere E., Jagadeesh J., Felgentreff K., Dobbs K., Al-Herz W., Jones M., Chandrasekharappa S., Kirby M., Wincovitch S., Simon K.L., Itan Y., DeVine A., Schlaeger T., Schambach A., Sood R., Notarangelo L.D, & Candotti F. (2015). Reticular dysgenesis–associated AK2 protects hematopoietic stem and progenitor cell development from oxidative stress. The Journal of Experimental Medicine, 212(8), 1185-1202.

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Transverse Aortic Constriction in ALK1 Mice

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Animals were treated in compliance with the Guide for the Care and Use of Laboratory Animals (National Academy of Science). Animal protocols were approved by the Tufts Medical Center Institutional Animal Care and Use Committee. Adult, male, 12–14 week old wild-type (Alk1^+/+) and Alk1^+/− mice underwent transverse aortic constriction (TAC) for 2 weeks using a 27G needle to generate left ventricular (LV) pressure overload [4 (link), 12 (link)]. Sham-operated mice served as controls (n=6/group). At the conclusion of the studies, terminal hemodynamics were recorded using biventricular conductance catheters as previously described [12 (link)]. LV tissue was rinsed with sterile saline and stored for subsequent biochemical and histological analysis. Alk1^+/− mice were obtained from The Jackson Laboratory. Genotyping of Alk1^+/− and Alk1^+/+ mice was performed according to the PCR protocol recommended by the The Jackson Laboratory using REDExtract-N-Amp PCR ReadyMix (Sigma-Aldrich). The primers used for genotyping were wildtype Alk1 (TCT GAC TGT AAG TAG TCT GGC TCA G), mutant Alk1 (GGG TGG GAT TAG ATA AAT GCC TGC TCT), and a common primer (CCT GTT CAG ATG CCT TCA GGA TGA G).

Morine K.J., Qiao X., Paruchuri V., Aronovitz M.J., Mackey E.E., Buiten L., Levine J., Ughreja K., Nepali P., Blanton R.M., Oh S.P., Karas R.H, & Kapur N.K. (2017). Reduced Activin Receptor Like-Kinase 1 Activity Promotes Cardiac Fibrosis in Heart Failure. Cardiovascular pathology : the official journal of the Society for Cardiovascular Pathology, 31, 26-33.

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Whole-mount in situ hybridization for zrsr2

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To generate a template for zrsr2 riboprobe synthesis for WISH, we performed RT-PCR using RNA from 5 dpf WT embryos and primers from the region shared by both isoforms (Supplementary Table S1). The RT-PCR product was cloned into a pCR4-TOPO vector (Thermo Fisher Scientific, Waltham, MA, USA) and sequence-verified by Sanger sequencing. The plasmid DNA was extracted using QIAprep Spin Miniprep Kit (Qiagen, Germantown, MD, USA), digested with SpeI, and labeled with a DIG labeling kit (Sigma-Aldrich, St. Louis, MO, USA) using T7 polymerase. Antisense riboprobes for all other WISH markers used in this study were also generated using a DIG labeling kit (Sigma-Aldrich, St. Louis, MO, USA). Zrsr2^hg129/+ fish were in-crossed, and embryos were collected at various developmental stages. Collected embryos were euthanized and fixed overnight in 4% PFA at 4 °C. WISH was performed as described previously [44 (link)]. For o-dianisidine staining, euthanized embryos were incubated in o-dianisidine (MP Biomedicals, Costa Mesa, CA, USA) solution for 15 min in the dark [45 (link)], followed by fixation in 4% PFA prior to imaging. Embryos used for WISH and o-dianisidine staining were genotyped via fluorescent PCR, as described previously [41 (link)], using REDExtract-N-Amp PCR ReadyMix (Sigma-Aldrich, St. Louis, MO, USA) for product amplification.

Weinstein R., Bishop K., Broadbridge E., Yu K., Carrington B., Elkahloun A., Zhen T., Pei W., Burgess S.M., Liu P., Bresciani E, & Sood R. (2022). Zrsr2 Is Essential for the Embryonic Development and Splicing of Minor Introns in RNA and Protein Processing Genes in Zebrafish. International Journal of Molecular Sciences, 23(18), 10668.

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Multiplex PCR Identification of E. coli

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Bacteria were grown from glycerol stocks on Tryptone Soya Agar (TSA) (Oxoid, Basington, UK) plate overnight at 37 ± 1 °C. Genomic DNA was extracted using a commercial kit (DNeasy Blood and Tissue Kit, Qiagen, Hilden, Germany), following the manufacturer’s instruction.
A multiplex PCR targeting four genes (lacY, lacZ, uidA, cyd) was used for E. coli identification, following the method described by Horakova et al. (2008) [115 (link)].
The PCR amplification was performed in a reaction volume of 10 µL containing 5 µL REDExtract-N-Amp PCR ReadyMix (Sigma-Aldrich, St Louis, MO, USA), 0.25 µL primers (10 pmol) and 1.5 µL DNA.
The following amplification parameters were applied: initial denaturation at 94 °C for 3 min, 30 cycles of denaturation at 94 °C for 30 s, annealing at 58 °C for 25 s, elongation at 72 °C for 30 s and a final extension at 72 °C for 3 min.
The amplified products were loaded onto a 2% agarose gel containing Syber Safe DNA Gel Stain (Invitrogen, Carlsbad, CA, USA) and run in 1X TBE buffer at 100 V for 1 h.
PCR fragments were visualized with a UV transilluminator. A pUC19 DNA/MspI (Hpall) Marker (Thermo Fisher Scientific, Waltham, MA, USA) was loaded on each gel as a DNA size standard. E. coli ATCC 25,922 DNA was present in every run as a positive control strain. Strains showing PCR products of 463 bp, 393 bp, 319 bp and 264 bp were considered E. coli.

Massella E., Reid C.J., Cummins M.L., Anantanawat K., Zingali T., Serraino A., Piva S., Giacometti F, & Djordjevic S.P. (2020). Snapshot Study of Whole Genome Sequences of Escherichia coli from Healthy Companion Animals, Livestock, Wildlife, Humans and Food in Italy. Antibiotics, 9(11), 782.

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Quantitative Analysis of Cardiac Cell Gene Expression

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Total cellular RNA was extracted and isolated from intact HD-CPCGs by TRIzol (Invitrogen, Carlsbad, CA) followed by preparation of cDNA using the SuperScript First-Strand Synthesis System (Invitrogen, Carlsbad, CA). RNA quantification and purity was determined by spectrophotometry. PCR was performed using REDExtract N-Amp PCR Ready Mix (Sigma-Aldrich, St. Louis, MO) for 30 cycles in a DNA thermocycler (Eppendorf, Hauppauge, NY) using specific primers for integrin receptor components alpha1, alpha2, alpha5, beta1, CD44, and for hyaluronan-mediated motility (RHAMM), alpha-smooth muscle actin (alphaSMA); and growth factors: bFGF, PDGF, VEGF, and SDF-1alpha. The PCR products were analyzed by gel electrophoresis and densitometry was measured using Image J analysis software (http://rsbweb.nih.gov/ij), and standardized to beta-actin.

Hilton S.A., Dewberry L.C., Hodges M.M., Hu J., Xu J., Liechty K.W, & Zgheib C. (2019). Mesenchymal stromal cells contract collagen more efficiently than dermal fibroblasts: Implications for cytotherapy. PLoS ONE, 14(7), e0218536.

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Conditional Cardiac-Specific Lmna Knockout

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Generation and phenotypic characterization of the Myh6-Cre:Lmna^F/F mice have been published.¹⁷ (link) In brief, to delete the Lmna gene in cardiac myocytes, the mice with the floxed exon 2 of the Lmna gene (Lmna^F/F) were crossed to the Myh6-Cre transgenic mice, as published.17 (link), 18 (link), 19 (link) Mice were housed in a 12-hour light/dark cycle facility with food and water available ad libitum.
Genomic DNA was extracted from tail clips and used for genotyping. In brief, the mouse tails were incubated with 50 μL extraction buffer and 12.5 μL tissue preparation solution (Sigma, cat. nos. E7526 and T3073) on a 55 °C water bath for 10 minutes followed by incubation at 95 °C for 3 minutes. Neutralization solution was added to the samples before polymerase chain reaction (PCR). An aliquot of 2 μL of the solution containing tail DNA from each sample was used along with REDExtract-N-AMP PCR Ready Mix (Sigma, cat. no. R4775) for genotyping. The list of the oligonucleotide primers used for genotyping is presented in Supplemental Table 1.

Cheedipudi S.M., Asghar S, & Marian A.J. (2022). Genetic Ablation of the DNA Damage Response Pathway Attenuates Lamin-Associated Dilated Cardiomyopathy in Mice. JACC: Basic to Translational Science, 7(12), 1232-1245.

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