Peripheral blood in ethylene diamine tetraacetic acid-containing (EDTA) collection tubes was used to extract genomic DNA (standard phenol-cloroform method). DNA was amplified using the polymerase chain reaction (PCR): PCR mix (50 μL) contained 200 ng of genomic DNA, 1.5 μL of 10 pmol primers, 5 μL of buffer 10x, 1.5 μL of MgCI2 50 mmol, 1 μL of 40 mmol dNTP and 2.5 U of Taq polymerase enzyme (Invitrogen Corp., Carlsbad, CA, USA). PCR products were analyzed on SYBR-Safe 3% agarose gel and displayed to the ultraviolet lamp. PCR products were sequenced bidirectional directly using Big-Dye terminator 3.1 cycle sequencing kit and run on ABI PRISM 3130 DNA analyzer (Applied Biosystems, Foster City, CA). Primers used for PCR and sequencing were designed in our laboratory (Table 2 ). Mutations in KLF-1 were detected by DNA sequencing of exon 2; mutations of BCL11A were found by sequencing of the 5 exons and the two intronic regions containing SNP rs11886868 and rs4671393. For the analysis of GATA1 gene the entire codifying region (6 exons) was sequenced. Also the XmnI genotype was obtained by XmnI enzymatic restriction. The PCR conditions will be made available upon request.
Taq polymerase enzyme
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
Taq polymerase is a DNA polymerase enzyme derived from the thermophilic bacterium Thermus aquaticus. It is a commonly used enzyme in polymerase chain reaction (PCR) for the amplification of DNA sequences.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Myod plasmid, by System Biosciences (2 mentions)
Abi prism 3130 dna analyzer, by Thermo Fisher Scientific (1 mentions)
Bigdye terminator 3.1 cycle sequencing kit, by Thermo Fisher Scientific (1 mentions)
Abi prism 7900ht sequence detection system, by Thermo Fisher Scientific (1 mentions)
Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
7 protocols using taq polymerase enzyme
1
DNA Extraction and Genetic Analysis Protocol
2
Regulation of miR-182 Promoter by MyoD
3
Regulation of miR-182 Promoter by MyoD
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Cells were transfected with different miR-182 promoter constructs with or without MyoD plasmid (lentiviral system from System Biosciences). Cells were harvested 36 h after transfection. Luciferase assays were performed as previously described (14 ). Plasmids with different miR-182 promoters were cloned into a luciferase vector (Promega). miR-182 promoter sequences were PCR amplified with high fidelity Taq polymerase enzyme (Invitrogen) using mouse genomic DNA as a template. Primer sequences are provided in Table S1. The amplified fragment was cloned into a Pgl3-basic vector at Kpn1 and Xho1 sites (Cho-Cho Cloning Kit). To mutate the putative MyoD binding sites in the miR-182 promoter, we adopted a two-step PCR ligation method with two sets of overlapping primers (Table S1) that span the new binding site with mutations. The two amplified PCR fragments were used as a template for the second PCR using primers Mir-182-pro-5.4 -kpn1 and Mir-182-pro-3.1-rev-xho1 and cloned using a similar strategy as described above.
4
Mutagenic Analysis of miR-182 Promoter
5
STR Genotyping of Canine Samples
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PCR reactions were performed to amplify the loci using 10 STR markers: Cun01, Cun05B, Cun08, Cun09, Cun10A, Cun11, Cun14, Cun16, Cun21B and Cun22. The primer pairs for these markers were described by Seyoum et al. [28 (link)]. The PCR reaction mixtures were prepared with the following reagents and concentrations to a final volume of 25 μL: 50 ng of DNA, 2.5 μL of 10× buffer (100 mM Tris-HCl, pH 8.3, 500 mM KCl), 1 μL of 15 mM MgCl2, 2 μL of the dNTPs mixture (0.2 mM each of dATP, dCTP, dGTP, and dTTP), 1 μM of each primer (20 μg/μL), 1.25 μL of Taq Polymerase enzyme (Invitrogen), and H2O to complete the final volume. The PCR reactions were carried out in a thermal cycler with an initial denaturation step at 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 45 s, annealing at a temperature of 58–60 °C (depending on the primer pair) for 90 s, and extension at 72 °C for 7 min.
6
WSSV Amplification from Multiple Loci
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Amplification of the WSSV from the 3 loci was carried out as described by Wongteerasupaya et al. (2003) (for ORF 94) and Pradeep et al. (2008) (for ORFs 75 and 125) (Table 2). Amplifications were prepared in 50 µl volumes with the following final concentrations: 0.2 mM dNTPs, 4 ng µl -1 of each oligonucleotide, 2.5 U of Taq polymerase enzyme (Invitrogen ® ), 1× buffer ], 50 mM KCl) MgCl 2 (1.5 mM for ORF 94; 2.0 mM for ORF 75 and ORF 125), and 1 µl of DNA. Final volume was adjusted with ddH 2 O. The cycling programs run in a gradient thermal cycler (Eppendorf ® ) are listed in Table 2. PCR pro ducts were resolved by electrophoresis in 1% agarose gels containing ethidium bromide, and they were visualized by UV transillumination.
7
Bacterial 16S rRNA Sequencing Protocol
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Isolates were identified by sequencing of the 16S rRNA genes amplified by PCR using primers 27F and 1492R (Lane 1991) . PCR reactions were performed in a total volume of 50 μl which contained 10 μmol l -1 of each primer (Invitrogen Life Technologies, Paisley, UK), 0.2 mmol l -1 dNTPs, 1U Taq polymerase enzyme (Invitrogen Life Technologies, Paisley, UK) and biomass was taken from a single colony as template.
PCR conditions were as follows: initial denaturation at 95°C for 5 min, 35 cycles of 95°C for 1min, 55°C for 1 min, 72°C for 1.5 min, a final elongation step at 72°C for 5 min. PCR products were cleaned up (QIAquick PCR purification kit, Qiagen, UK), quantified using NanoDrop ND-1000 spectrophotometer (Thermo Scientific, DE, USA) and sequenced with primers 27F and 1492R using BigDye Terminator v3.1 cycle sequencing kit and ABI Prism 7900HT sequence detection system (Applied Biosystems, UK). The sequences were assembled using SeqMan, DNA Star Lasergene 2.0 and analysed using BLAST at http://blast.ncbinlm.nih.gov/Blast.cgi (Altschul et al. 1990 ). Sequences were aligned and a neighbour-joining phylogenetic tree was constructed using ARB (Ludwig et al. 2004) .
PCR conditions were as follows: initial denaturation at 95°C for 5 min, 35 cycles of 95°C for 1min, 55°C for 1 min, 72°C for 1.5 min, a final elongation step at 72°C for 5 min. PCR products were cleaned up (QIAquick PCR purification kit, Qiagen, UK), quantified using NanoDrop ND-1000 spectrophotometer (Thermo Scientific, DE, USA) and sequenced with primers 27F and 1492R using BigDye Terminator v3.1 cycle sequencing kit and ABI Prism 7900HT sequence detection system (Applied Biosystems, UK). The sequences were assembled using SeqMan, DNA Star Lasergene 2.0 and analysed using BLAST at http://blast.ncbinlm.nih.gov/Blast.cgi (Altschul et al. 1990 ). Sequences were aligned and a neighbour-joining phylogenetic tree was constructed using ARB (Ludwig et al. 2004) .
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