For DNA extraction, a bacterial suspension was prepared in 500 μl of 0.2 mM EDTA, 30 μl of 1 M NaOH was added, and the sample was boiled for 5 min. After 2 min centrifugation at 16.000 x g, the supernatant was recovered. PCR amplification was performed with a DNA thermocycler (Eppendorf). Each reaction mixture contained 5 μl of PCR buffer (EG Healthcare) and 5 μl of each of the four deoxynucleoside triphosphates (Roche) at a final concentration of 200 μM each. A total of 2.5 μl of each primer was used at a concentration of 10 μM, with 5 U of Taq DNA polymerase (EG Healthcare), in a total volume of 50 μl. The cycling conditions for the rpoD (PsEG30F/PsEG790R) [18 (link)], 16S rRNA (16F27/16R1492) [23 ] and gyrB (BAUP2/APrU) genes [24 (link)] included a denaturation period at 94°C for 5 min, followed by 30 cycles of amplification (denaturation at 94°C for 1 min, primer annealing at 55°C (48°C for rpoD) for 1 min, and primer extension at 72°C for 1.5 min). A final elongation step was carried out at 72°C for 10 min. The amplified products were purified with MultiScreen HTS PCR 96-well filter plates (Millipore) according to the manufacturer’s instructions. Sequencing reactions were performed using ABI Prism BigDye Terminator version 3.1, and the sequences were read with an automatic sequence analyzer (3130 genetic analyzer; Applied Biosystems).
Dna thermocycler
Manufactured by Eppendorf
Sourced in Germany
The DNA thermocycler is a laboratory instrument used for the amplification of DNA samples. It precisely controls the temperature and duration of the thermal cycling process, which is a crucial step in various molecular biology techniques, such as polymerase chain reaction (PCR).
Automatically generated - may contain errors
Lab products found in correlation
Abi prism bigdye terminator version 3, by Thermo Fisher Scientific (1 mentions)
3130 genetic analyzer, by Thermo Fisher Scientific (1 mentions)
Redextract n amp pcr readymix, by Merck Group (1 mentions)
Superscript first strand synthesis system, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
5 protocols using dna thermocycler
1
Bacterial DNA Extraction and Amplification
2
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.
3
HPV16/18 Detection via β2-Microglobin PCR
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To determine the absence of PCR inhibitors and the integrity of the DNA, a 165 bp segment of a human β2-microglobin gene was amplified. HPV16/18 DNA was analyzed only for those samples that were positive for the β2-microglobin sequence. Primer pairs located in the E6 gene open reading frame (ORF) which are specific for HPV subtype were used. 1.0 µg of DNA in a total volume of 25 µL (1× Tris buffer, 1.5 mM MgCl2, 1 µM of each primer, 200 µM each of dATP, dCTP, dGTP, dTTP and 1 U Taq) polymerase was used to perform the amplification reactions. In the DNA thermocycler (Eppendorf) 35 amplification cycles were performed with the following profile:
The last cycle was followed by a final extension of 72 °C for 7 minute. A negative control (Sterile water instead of DNA) and positive control (HPV16 positive Cervical and HPV 18 positive uterine carcinoma) was used during each PCR experiments. 1.5% agarose gel electrophoresis and ethidium bromide staining were used to analyze the PCR products. SPSS 17.0 version was used to analyze the resulting data.
The last cycle was followed by a final extension of 72 °C for 7 minute. A negative control (Sterile water instead of DNA) and positive control (HPV16 positive Cervical and HPV 18 positive uterine carcinoma) was used during each PCR experiments. 1.5% agarose gel electrophoresis and ethidium bromide staining were used to analyze the PCR products. SPSS 17.0 version was used to analyze the resulting data.
4
Actinobacterial 16S rDNA Amplification
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PCR primers were designed to amplify the 16S rDNA fragment from the genomic DNA of Streptomyces spp. as previously described (23) . The sequences and properties of the primers were: forward:
5'-GACAAGCCCTGGAAACGGGGT-3' and reverse 5'-GCTCGTGTCGTGAGATGTTGGG-3', synthesized by Macrogen (Seoul, South Korea). PCR was performed using a DNA thermocycler (Eppendorf, Hamburg, Germany) to amplify the targeted 16S rDNA fragments (~ 900 bp) of the soil isolated Actinomycetes. The reactions were performed in a final volume of 30 µL including 10 µL of Readyto-use PCR mixture (prepared following manufacturer protocol EasyTaq® PCR SuperMix (2×)), 1.5 µL of forward primer (10 pmol/μL), 1.5 µL of reverse primer (10 pmol/μL), 3 µL of genomic DNA template, 14 µL of ddH2O. The program was performed as follows: initial denaturing at 95°C for five min, 35 cycles of denaturation at 95°C for 30 sec, annealing at 60°C for 40 sec, extension at 72°C for 45 sec, and final extension at 72°C for 3 min.
5'-GACAAGCCCTGGAAACGGGGT-3' and reverse 5'-GCTCGTGTCGTGAGATGTTGGG-3', synthesized by Macrogen (Seoul, South Korea). PCR was performed using a DNA thermocycler (Eppendorf, Hamburg, Germany) to amplify the targeted 16S rDNA fragments (~ 900 bp) of the soil isolated Actinomycetes. The reactions were performed in a final volume of 30 µL including 10 µL of Readyto-use PCR mixture (prepared following manufacturer protocol EasyTaq® PCR SuperMix (2×)), 1.5 µL of forward primer (10 pmol/μL), 1.5 µL of reverse primer (10 pmol/μL), 3 µL of genomic DNA template, 14 µL of ddH2O. The program was performed as follows: initial denaturing at 95°C for five min, 35 cycles of denaturation at 95°C for 30 sec, annealing at 60°C for 40 sec, extension at 72°C for 45 sec, and final extension at 72°C for 3 min.
5
Molecular Identification of Fungal Isolates
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The molecular identification of the isolates was done using ITS primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS2 (5'-GCTGCGTTCTTCATCGATGC-3'). Polymerase Chain Reaction (PCR) was carried out in flat capped 200 μL volume PCR tubes obtained from M/s Tarsons, Kolkata, India. PCR amplification of the partial region of ITS was performed in a 20 µl reaction mix containing 5X reaction buffer, 25 mM MgCl2, 0.01 mM dNTPs, 0.001 M primers, 1 U of TaqDNA polymerase (Genei) and 100 ng DNA total. PCR amplifications were performed on a DNA thermocycler (Eppendorf) with the following program: an initial denaturation at 97 0 C for 6 min followed by 35 amplification for cycles of 94 0 C for 1 min, 58 0 C for 1 min, 72 0 C for 2 min followed by final extension at 72 0 C for 7 min.
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