Phire animal tissue direct pcr kit

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Phire Animal Tissue Direct PCR Kit is a reagent kit designed for fast and direct PCR amplification from animal tissue samples without prior DNA extraction. The kit provides a simple and efficient protocol for direct PCR from a variety of animal tissues.

Automatically generated - may contain errors

Lab products found in correlation

Dneasy blood and tissue kit, by Qiagen (2 mentions) Primestar gxl dna polymerase, by Takara Bio (2 mentions) Lysozyme m lysm cre mice, by Jackson ImmunoResearch (1 mentions) Gli3fl fl mice, by Jackson ImmunoResearch (1 mentions) β actin, by Promega (1 mentions) T7 endonuclease 1, by New England Biolabs (1 mentions) F12 glutamax, by Thermo Fisher Scientific (1 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions) C1000 touch thermal cycler, by Bio-Rad (1 mentions) Chromas software, by Technelysium (1 mentions) Kcal fat diet, by Research Diets (1 mentions) C57bl 6 mice, by Orient Bio (1 mentions) Megascript t7, by Thermo Fisher Scientific (1 mentions) R1016, by Zymo Research (1 mentions) Rneasy mini kit, by Qiagen (1 mentions) Np 40, by Merck Group (1 mentions) Tergitol np 40, by Merck Group (1 mentions) Crispr cas9 enzyme, by PNA Bio (1 mentions) Megascript t7 transcription kit, by Thermo Fisher Scientific (1 mentions) Quantitect sybr green pcr kit, by Qiagen (1 mentions)

38 protocols using phire animal tissue direct pcr kit

Conditional Deletion of Gli3 in Myeloid Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

Gli3^fl/fl mice and lysozyme M (LysM)-cre mice were obtained from Jackson Labs (Bar Harbor, ME). Mice were bred and maintained at the UNH animal resource office (ARO). Mice were crossed to generate mice with conditional deletion of Gli3 in myeloid cells (M-Gli3^−/−). Mice were genotyped at p6-p9 using toe clipping and were separated into M-Gli3^−/− or WT groups. All experiments were approved by the UNH IACUC following guidelines of the National Institutes of Health. Mice were used for experiments when they reached 8–14 weeks of age.
Genotyping was performed using DNA extracted from mice toes using Phire Animal Tissue Direct PCR Kit (Fisher Scientific, F-140WH). The following PCR primer pairs were used: Gli3 flox, 5′-GATGAATGTGATCCAGGGC-3′ (forward) and 5′- GTCATATTGTGCCCAGTAGTAGC-3′ (reverse); Cre, 5′-GCGGTCTGGCAGTAAAAACTATC-3′ (forward) and 5′-GTGAAACAGCATTGCTGTCACTT-3′ (reverse); and recombined Gli3 (rGLI3), 5′-CTGGATGA ACCAAGCTTTCCATC-3′ (forward) and 5′-CAGTA GTAGCCTGGTTACAG-3′ (reverse). Samples were analyzed on 2% agarose gels.

Matissek S.J., Karbalivand M., Han W., Boutilier A., Yzar-Garcia E., Kehoe L.L., Gardner D.S., Hage A., Fleck K., Jeffers V., Rajsbaum R, & Elsawa S.F. (2022). A novel mechanism of regulation of the oncogenic transcription factor GLI3 by toll-like receptor signaling. Oncotarget, 13, 944-959.

+ Open protocol

+ Expand

Genotyping Mice Genes by PCR

Check if the same lab product or an alternative is used in the 5 most similar protocols

Genomic DNA from tail snips were isolated either by boiling in NaOH or using the Phire® Animal Tissue Direct PCR Kit (Fisher Scientific Pittsburgh, PA) according to manufacturer's instructions. The genotype of each gene was confirmed by PCR: Chop, [40] , GHL⁺[9] (link), Ask1^−/−[32] (link), SOX21 (Phire® Animal Tissue Direct PCR Kit) and β-actin (Promega, Madison, WI) were used as loading controls.

Adekeye A., Haeri M., Solessio E, & Knox B.E. (2014). Ablation of the Proapoptotic Genes Chop or Ask1 Does Not Prevent or Delay Loss of Visual Function in a P23H Transgenic Mouse Model of Retinitis Pigmentosa. PLoS ONE, 9(2), e83871.

+ Open protocol

+ Expand

Cardiac Gene Editing in Neonatal Mice

Check if the same lab product or an alternative is used in the 5 most similar protocols

Neonatal mice were genotyped at postnatal day 3 (P3) using the Phire Animal Tissue Direct PCR Kit (Fisher Scientific) according to manufacturer's instructions. At P3 or P10, heterozygous (Myh6 Cas9+/-) or homozygous (Myh6 Cas9+/+ ) Myh6 Cas9 mice or their littermate controls (Myh6-Cre negative) were injected with AAV9-sgRNA at either a dose of 5×10 11 (low dose), 1×10 12 (link) (medium dose), or 2.5×10 12 (link) (high dose) viral genomes per animal by intraperitoneal injection. For dual sgRNA delivery, a medium dose was used (this was the maximum dose that could be achieved with the viral titer obtained). Adult mice (8 weeks) were anesthetized with a low dose mixture of ketamine (60 mg/kg) and xylazine (7 mg/kg) by intraperitoneal injection and intubated with a tracheal tube connected to a ventilator. Mice were supplemented and maintained under anesthesia with 1.5% isoflurane. A surgical plane of anesthesia was confirmed by a lack of a pain reflex. The free wall of the left ventricle was injected with a high dose of AAV9-sgSav1 in 2 locations (total volume per injection was 6 μL). The muscle and rib cage were closed with 5-0 silk suture, and analgesia (buprenorphine, 0.05-0.1 mg/kg) were given immediately after surgery and as necessary.

Johansen A.K., Molenaar B., Versteeg D., Leitoguinho A.R., Demkes C., Spanjaard B., de Ruiter H., Akbari Moqadam F., Kooijman L., Zentilin L., Giacca M, & van Rooij E. (2017). Postnatal Cardiac Gene Editing Using CRISPR/Cas9 With AAV9-Mediated Delivery of Short Guide RNAs Results in Mosaic Gene Disruption. Circulation research, 121(10).

+ Open protocol

+ Expand

CRISPR/Cas9 Genome Editing in Embryos

Check if the same lab product or an alternative is used in the 5 most similar protocols

CRISPR/Cas9 mixes were microinjected into embryos less than 1 h old. Embryos that had not been injected were used as controls. After 24 h, embryos were pooled in groups of 20–30 and genomic DNA was isolated using the Phire Animal Tissue Direct PCR kit (#F140WH, ThermoFisher Scientific). PCR was performed to amplify 857 bp of the DNA region containing the shibire mutation loci using the Phire polymerase with the forward primer 5′-CACCAGTTTCGATGAGATCC-3′ and reverse primer 5′ CAAAGCTGAACCGGAACCTT-3′. The PCR cycling conditions used were 98 °C for 5 min, 35 cycles of 98 °C for 5 s, 60 °C for 5 s and 72 °C for 40 s, followed by a final extension at 72 °C for 1 min. 200 ng of PCR amplicon were then denatured and reannealed using the following conditions: 95 °C for 5 min, ramp down to 85 °C at − 2 °C/s and ramp down to 25 °C at − 0.1 °C/s. The annealed PCR products were then incubated with 1 μL of T7 endonuclease I (New England BioLabs, M0302S) at 37 °C for 15 mins. Products were visualised using 2% agarose (Scientifix Pty. Ltd., #9010E) gel electrophoresis.

Choo A., Fung E., Chen I.Y., Saint R., Crisp P, & Baxter S.W. (2020). Precise single base substitution in the shibire gene by CRISPR/Cas9-mediated homology directed repair in Bactrocera tryoni. BMC Genetics, 21(Suppl 2), 127.

+ Open protocol

+ Expand

Alzheimer's Transgenic Mice Exercise Study

Check if the same lab product or an alternative is used in the 5 most similar protocols

Generation of male Alzheimer-transgenic (B6C3-Tg(APPswe,PSEN1dE9)85Dbo/J) mice were used in the experiments. Three-month-old wild type (WT) (no transgenic modulation without any training) (n = 5), Alzheimer transgenic mice (AD) (n = 5) and trained Alzheimer disease (TAD) mice (n = 5) were kept under light/dark cycles of 12/12 h with free access to food and water. Alzheimer transgenic mice were trained on treadmill four times per week for one hour divided into 10 sessions. One session contained 2 min low intensity running (10 m/min) and 4 min high intensity running (20 m/min). The study was carried out in accordance with ethical guidelines (ethical permission number for this study: PEI/001/2105-6/2014 (09.07.2014), Semmelweis University, Hungary). Genotyping was performed using Phire Animal Tissue Direct PCR Kit (Thermo Fischer Scientific, Waltham, MA, USA) according to the manufacturer’s instructions.

Szegeczki V., Horváth G., Perényi H., Tamás A., Radák Z., Ábrahám D., Zákány R., Reglodi D, & Juhász T. (2020). Alzheimer’s Disease Mouse as a Model of Testis Degeneration. International Journal of Molecular Sciences, 21(16), 5726.

+ Open protocol

+ Expand

Alzheimer's disease progression and exercise

Check if the same lab product or an alternative is used in the 5 most similar protocols

Male Alzheimer transgenic [B6C3-Tg(APPswe,PSEN1dE9)85Dbo/J] (n = 5) mice were used to follow the effects of AD. Three-month-old wild type (WT; no transgenic modulation and no training, n = 5), Alzheimer transgenic mice (AD, n = 5), and trained Alzheimer’s disease mice (TAD, n = 5) were kept under light/dark cycles of 12:12 h with food and water ad libitum. Alzheimer transgenic mice were trained on a treadmill four times per week for 1 h divided into 10 sessions. One session contained 2 min of low-intensity running (10 m/min) and 4 min of high-intensity running (20 m/min). The study was carried out in accordance with ethical guidelines (ethical permission number: PEI/001/2105-6/2014, Semmelweis University, Hungary). Genotyping was performed using a Phire Animal Tissue Direct PCR Kit (Thermo Fischer Scientific, Waltham, MA, United States) according to the manufacturer’s instructions.

Perényi H., Szegeczki V., Horváth G., Hinnah B., Tamás A., Radák Z., Ábrahám D., Zákány R., Reglodi D, & Juhász T. (2020). Physical Activity Protects the Pathological Alterations of Alzheimer’s Disease Kidneys via the Activation of PACAP and BMP Signaling Pathways. Frontiers in Cellular Neuroscience, 14, 243.

+ Open protocol

+ Expand

Genotyping Mice for Cre and IGF1R Alleles

Check if the same lab product or an alternative is used in the 5 most similar protocols

Genomic DNA was extracted from tail snips and other tissues (spleen, muscle, lung, heart, gut, liver, growth plate cartilage, callas and bone) of the mice using DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA, USA) for purification and Phire Animal Tissue Direct PCR Kit for amplification (Thermo Fisher Scientific Inc., Marietta, OH, USA). Polymerase chain reaction (PCR) analyses of the DNA were performed to detect Cre and floxed-IGF1R alleles using corresponding primer sets with standard conditions (5 min at 98°C; 5 s at 98°C, 5 s at 61°C, and 20 s at 72°C for 37 cycles; 1 min at 72°C; ∞ at 4°C). PCR was performed with a mixture of three primers (two forward primers, 5′-CTT CCC AGC TTG CTA CTC TAG G-3′ and 5′-TGA GAC GTA GCG AGA TTG CTG TA-3′, and a reverse primer, 5′-CAG GCTTGC AAT GAG ACA TGG G-3′) to detect 320-bp and 120-bp products from the non-excised and excised gene alleles, respectively.^{(18 (link))} The Cre transgene was detected by PCR using the primers 5′-GCA AAA CAG GCT CTA GCG TTC G-3′ (forward) and 5′-CTG TTT CAC TAT CCZ GGT TAC GG-3′ (reverse) to amplify a 560-bp DNA product. Cre-positive and gene excision (Δ-IGF1R) positive littermates were designated as experimental groups whereas Cre-negative littermates were used as controls.

Wang T., Wang Y., Menendez A., Fong C., Babey M., Tahimic C.G., Cheng Z., Li A., Chang W, & Bikle D.D. (2015). Osteoblast-Specific Loss of IGF1R Signaling Results in Impaired Endochondral Bone Formation During Fracture Healing. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research, 30(9), 1572-1584.

+ Open protocol

+ Expand

TALEN-Mediated Genomic Modification in Rat C6 Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

10⁵ rat C6 cells were cultured in F-12 GlutamaX (Invitrogen, now Life Technologies), 10% FBS heat-inactivated and seeded in 6 well plates. Cells were transfected the next morning with plasmids encoding for TALENs at 0.6 µg total DNA per well and Lipofectamine 2000 (Invitrogen, now Life Technologies) following manufacturer's instructions. They were kept at 37°C for 4 hours in F-12 medium without FBS and Lipofectamine 2000. Then the medium was replaced by complete medium (F-12 and FBS), and cells were left for 72 hours at 30°C. Then cells were harvested and genomic DNA was extracted using Phire Animal Tissue Direct PCR Kit (Thermo Scientific) and amplified. Primers for amplification are listed in Table S2 in File S1. 5 µl of the PCR mix were heated at 95°C for 10 minutes and then the temperature was decreased by 5°C every minute until it reached 10°C in a Thermocycler.

Ponce de León V., Mérillat A.M., Tesson L., Anegón I, & Hummler E. (2014). Generation of TALEN-Mediated GRdim Knock-In Rats by Homologous Recombination. PLoS ONE, 9(2), e88146.

+ Open protocol

+ Expand

PCR-based DNA Lesion Verification

Check if the same lab product or an alternative is used in the 5 most similar protocols

For verification in DNA lesions at the intended Antp-Ubx_BE site, a pupal wing fragment harboring visible mutant clones (Figure 4—figure supplement 3B”) or control wild-type tissue were PCR amplified using the diluted protocol of the Phire Animal Tissue Direct PCR Kit (ThermoFisher) and a pair of oligonucleotides (Forward: 5’-ACCGATCGTAAACGTCAACTTAACG-3’; Reverse 5’-TACTGCGGTGGCGAGTGAATG-3’), before purification and Sanger sequencing using the reverse primer.

Tendolkar A., Mazo-Vargas A., Livraghi L., Hanly J.J., Van Horne K.C., Gilbert L.E, & Martin A. (2024). Cis-regulatory modes of Ultrabithorax inactivation in butterfly forewings. eLife, 12, RP90846.

+ Open protocol

+ Expand

Genotyping of CRISPR-Edited Liverpool Embryos

Check if the same lab product or an alternative is used in the 5 most similar protocols

Genomic DNA was extracted from non-injected or sgRNA-injected Liverpool embryos following the Nucleospin Tissue kit protocol (Machery-Nagel, Bethlehem, PA). Embryo assays were performed with the LightScanner Master Mix kit (Idaho Technology Inc., Salt Lake City, UT), and mutant detection was performed on adult legs with the Phire Animal Tissue Direct PCR kit (Thermo Fisher Scientific, Waltham, MA) with added LCGreen Plus+ Melting dye (Idaho Technology Inc., Salt Lake City, UT). All samples were amplified with the C1000 Touch Thermal Cycler (Bio-rad, Hercules, CA) before being analyzed with the LightScanner Call-IT 2.0 software on the LightScanner instrument (Idaho Technology Inc., Salt Lake City, UT). Sanger sequencing was performed at the Laboratory for Genomic Technologies (Institute for Plant Genomics and Biotechnology, Texas A&M University, College Station, TX) and chromatograms were analyzed using Chromas software (Technelysium, Australia). A list of primers used are located in S1 Table.

O’Leary S, & Adelman Z.N. (2020). CRISPR/Cas9 knockout of female-biased genes AeAct-4 or myo-fem in Ae. aegypti results in a flightless phenotype in female, but not male mosquitoes. PLoS Neglected Tropical Diseases, 14(12), e0008971.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!