The (TTTAT)3-5 microsatellite polymorphism within intron 6 of the LDHA gene was analysed using conventional PCR reaction. PCR primers (Table 1 https://primer3.ut.ee/ ). PCR reaction mixtures contained ~80 ng of genomic DNA, 15 pmol of each primer, 1 x PCR buffer, 1.5 mM MgCl2, 200 μM dNTP, 0.4 unit of Taq polymerase (Eurx, Gdańsk, Poland) and nuclease-free water (AppliChem, Darmstadt, German) in a total volume of 15 μl. The PCR amplification program was as follows: 5 min initial denaturation at 94°C followed by 35 cycles (denaturation at 94°C for 30 s, primer annealing at 61°C for 30 s and product synthesis at 72°C for 30 s) and final elongation at 72°C for 5 min (Biometra T-Personal thermocycler, Biometra GmbH, Göttingen Germany). Finally, PCR products were separated by horizontal electrophoresis (80 V, 400 mA, 240 min; Mini-Sub Cell GT Systems, Bio-Rad, Hercules, CA, United States) on a 5% agarose gel (Norgen Biotek Corp., Canada) [4 ].
Nuclease free water
Manufactured by AppliChem
Sourced in Germany
Nuclease-free water is a high-purity water product specifically designed for use in molecular biology applications. It is free from detectable RNase, DNase, and other nucleases, ensuring the integrity of sensitive nucleic acid samples during handling and processing.
Automatically generated - may contain errors
Lab products found in correlation
Mini sub cell gt system, by Bio-Rad (1 mentions)
Biometra tpersonal thermocycler, by Analytik Jena (1 mentions)
Taq polymerase, by EURx (1 mentions)
Triazole reagent, by Thermo Fisher Scientific (1 mentions)
Nuclease free glycogen, by Thermo Fisher Scientific (1 mentions)
Isopropanol, by Merck Group (1 mentions)
Chloroform, by Merck Group (1 mentions)
Nucleospin bead tubes, by Macherey-Nagel (1 mentions)
Avian myeloblastosis virus reverse transcriptase, by Promega (1 mentions)
Dntp mix, by Thermo Fisher Scientific (1 mentions)
Mgcl2, by Vivantis Technologies (1 mentions)
Taq dna polymerase, by Vivantis Technologies (1 mentions)
T100 thermal cycler, by Bio-Rad (1 mentions)
5 protocols using nuclease free water
1
LDHA Gene Microsatellite Polymorphism Analysis
2
RNA Extraction from Serum Samples
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Serum samples were incubated on ice for a complete thaw process. Immediately, triazole reagent (1:1 ratio) (Thermo Fisher Scientific, MA, USA) and nuclease-free glycogen (1 μg/mL) (Thermo Fisher Scientific, MA, USA) were added, respectively. They were then incubated at room temperature for 10 min. Afterward, 200 μL of chloroform (Merck Millipore, Darmstadt, Germany) was added and the samples were incubated for 15 min. Next, they were centrifuged at 12,000 × g for 15 min at 4 °C. Upper aqueous phase was transferred, and 1.2 mL of isopropanol (Merck Millipore, Darmstadt, Germany) was added to the samples. After that, the samples were vortexed and then incubated for 10 min at room temperature. They were centrifuged at 12,000 × g for 8 min. The supernatants were aspirated carefully. One milliliter of 75% ethanol was added and the samples werecentrifuged at 7500 × g for 5 min at room temperature. The supernatant was carefully removed, and the tubes were dried out at room temperature. Each pellet was resuspended with 20 μL of nuclease-free water (AppliChem, Gatersleben, Germany) and stored at –80 °C for further evaluation.
3
Improved DNA/RNA Phenol Extraction Protocol
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For each sample, two reference specimen were subjected to DNA extraction using a slightly modified version of the improved DNA/RNA phenol extraction protocol by Griffiths et al. (2000 (link)) as proposed by Töwe et al. (2011 (link)). Briefly, samples were allowed to warm to room temperature and mixed thoroughly, followed by breakup of the cells by a 3 min vortex at 3200 rpm (Vortex Genie 2 labshaker, Scientific Industries, New York, USA) in NucleoSpin Bead Tubes (Macherey–Nagel, Düren, Germany). The obtained pellet was taken up in 150 µL of nuclease free water (AppliChem, Darmstadt, Germany). 20 µL aliquots were stored at 4 °C until analysis.
4
Total RNA Extraction and cDNA Synthesis
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Total RNA was isolated from 250 µl of supernatant using TRIrisol reagent according to the manufacturer's instructions (Invitrogen, Carlsbad, CA). Purified RNA was resuspended in 20 µl of sterile nuclease free water (AppliChem, Darmstadt, Germany). For cDNA synthesis, 2 µg of total RNA and 1 µl of 20 µM P-genotype-specific RT primer (TGG CTT CGC TCA TTT TAT AGA CA) or G-genotype-specific RT primer (TAG CTC CTT TTA ATG TAT GG) (final volume 10 µl) was incubated at 95°C for 5 min, chilled on ice and heated again for 5 min at 65°C. Actual reverse transcription was performed at 42°C for 60 min (final volume 26 µl) using 7 units of Avian Myeloblastosis Virus reverse transcriptase, according to the manufacturer's instructions (Promega, Madison, WI).
5
Investigating blaA and blaB Genes in Y. enterocolitica
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The blaA and blaB genes were investigated by PCR in all 18 Y. enterocolitica biovar 1A isolates from food. The blaA primer se-quences were A9-f (5'-GAG ATT CAG GAA TGA AGC ACT CTT CG-3') and A10-r (5'-TCA GGA TAT TTG CGA CAA AAT TAT-3'), which were predicted to yield an 896 bp product (2) . The blaB primer sequences were blaB5 (5'-CCC ACT TTA TAC CTT GGC ACA AA-3') and blaB3 (5'-GAA CAT ATC TCC TGC CTG GAA AT-3'), which were predicted to yield an 827 bp product (18) . All reactions were carried out with the T100 thermal cycler (Bio-Rad, Hercules, USA). PCR mixture (50 µL) contained 5 µl of 10× PCR buffer (Vivantis Technologies, Selangor DE, Malaysia), 4 mM MgCl 2 (Vivantis), 0.2 mM dNTP mix (Thermo Fisher Scientific, Waltham, Massachusetts, USA), 0.4 µM (each) primers (Biomers, Ulm, Germany), 1.5 U of Taq DNA polymerase (Vivantis), 5 µl of 50 ng of template DNA and 35.7 µl nuclease free water (AppliChem, Darmstadt, Germany). PCR reaction conditions included an initial denaturation (94ºC, 5 min), 30 cycles of denaturation (94ºC, 1 min), annealing (56ºC, 1 min), extension (72ºC, 1 min) followed by final extension (72ºC, 8 min). The products were analyzed by electrophoresis (Bio-Rad) in a 1% agarose gel and photographed using a UV transilluminator (DNR Minilumi Bio-imaging Systems Ltd., Jerusalem, Israel). Y. enterocolitica ATCC 23715 was used as a control in this study.
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