The MTL configuration (heterozygous or homozygous) was determined by RT-PCR using primers specific for MTLa and MTLα.[6 (link)] RT-PCR reactions were carried out on Bio-Rad T100 thermocycler (Bio-Rad, USA) using a standard program: 94°C incubation 5 min, and then 35 cycles of 94°C for 60 s, 55°C for 60 s, 72°C for 60 s, followed by a final extension step at 72°C for 10 min. Primer sequences were as follows: MTLa1 forward, 5’-TTGAAGCGTGAGAGGCAGGAG-3’ and MTLa1 reverse, 5’-GATTAGGCTGTTTGTTCTTCTCG-3’; and MTLα2 forward, 5’-CATGAATTCACGTCTGGAGGCAC-3’ and MTLα2 reverse, 5’-AAGCAGCCAACTCAGGTCAC-3’.
T100 thermocycler
Manufactured by Bio-Rad
Sourced in United States, China, Germany, United Kingdom
The T100 thermocycler is a laboratory instrument used for performing polymerase chain reaction (PCR) experiments. It provides precise temperature control and cycling for DNA amplification. The T100 thermocycler is capable of running a wide range of PCR protocols and is designed for reliable, consistent performance in the laboratory setting.
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Lab products found in correlation
Iscript cdna synthesis kit, by Bio-Rad (9 mentions)
Trizol reagent, by Thermo Fisher Scientific (8 mentions)
Cdna synthesis kit, by Promega (7 mentions)
Rneasy mini kit, by Qiagen (6 mentions)
Master mix pcr amplification reagent, by Promega (6 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (6 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (6 mentions)
Trizol, by Thermo Fisher Scientific (5 mentions)
Gotaq green master mix, by Promega (5 mentions)
M mlv reverse transcriptase, by Thermo Fisher Scientific (4 mentions)
Qiaquick pcr purification kit, by Qiagen (4 mentions)
Rneasy plus mini kit, by Qiagen (4 mentions)
Dneasy blood and tissue kit, by Qiagen (4 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (4 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Dnase 1, by Thermo Fisher Scientific (3 mentions)
Qiaquick gel extraction kit, by Qiagen (3 mentions)
Itaq universal sybr green supermix, by Bio-Rad (3 mentions)
Gelred, by Biotium (3 mentions)
Q5 high fidelity dna polymerase, by New England Biolabs (3 mentions)
202 protocols using t100 thermocycler
1
RT-PCR Determination of MTL Mating Type
2
Genetic Marker Analysis in Tomato
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PCR reactions were carried out according to the manufacturer’s instructions (SolGent Co., Ltd., Daejeon, Korea) in a total volume of 25 μl containing 1 μl genomic DNA, 2.5 μl 10X e-Tag reaction buffer, 0.5 μl of 10 mM dNTP mix, 1 μl of each forward and reverse primers, 0.125 μl Solg e-Taq DNA polymerase, and 18.875 μl of ddH2O. PCR amplification was carried out using a Bio-Rad T100 thermocycler (Bio-Rad Laboratories, Inc.) with the following conditions: denaturing for 3 min at 95°C, followed by 34 cycles of 30 s at 95°C denaturation, 30 s at annealing temperature (which varied for different primer sets (Table 3 )), 1 min at 72°C extension, and a final elongation step at 72°C for 5 min. PCR products were digested with the respective restriction enzymes. The reaction mixture consisted of 5 μl template PCR product, 1 μl reaction buffer, 0.1 μl of restriction enzyme, and 3.9 μl ddH2O. The mixture was incubated at 37°C for 16 hrs and was carried out using Bio-Rad T100 thermal cycler (Bio-Rad Laboratories, Inc.). The digested product was mixed with 2 μl 6X DNA loading buffer and subjected to gel electrophoresis on a 3% agarose gel to visualize the polymorphic DNA bands. The details of CAPS/dCAPS marker information, including primer sequences, restriction enzymes, and the expected band sizes for resistant and susceptible tomato groups, are presented in Table 3 .
3
Mycoplasma Detection in Bovine Samples
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All bovine field samples were inoculated onto Mycoplasma agar [Mycoplasma agar base (Oxoid CM0401); Milli-Q water; 0.2% w/v calf thymus DNA (Sigma D1501); Mycoplasma Selective Supplement G (Oxoid SR0059C); prepared by Elizabeth Macarthur Agricultural Institute (EMAI); NSW Department of Primary Industries, NSW, Australia] and incubated at 37°C in candle jars in elevated CO2 levels for 5 to 10 days. Following positive mycoplasma growth, several colonies from each sample were selected and placed in PBS for speciation by an ‘in house’ developed universal Mycoplasma spp. conventional PCR assay modified from Tang et al [20 ]. Reaction mixtures contained 0.25 mM dNTPs, 2.5 mM MgCl2, 1.5 U of GoTaq, 0.25 μM of each primer (Table 1 ), 8 μL of 5x Buffer and 5 μL DNA template in a final volume of 40 μL. Cycling conditions were 94°C for 5 min, followed by 35 cycles of 94°C for 30s, 55°C for 30s, 72°C for 1 min, and a final extension of 72°C for 5 min [28 (link)]. The assay was performed on a Bio-Rad-T100 Thermocycler (Bio-Rad Laboratories Pty Ltd, Gladesville, NSW, Australia). The PCR products from this assay were then speciated via Sanger Sequencing (Australian Genome Research Facility Ltd, Sydney, NSW, Australia).
4
Quantifying Chorionic Gene Expression in Fly Ovaries
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Total RNA from transgenic (control and s36-targeted) fly ovaries was extracted following a Trizol-based protocol (Molecular Research Center Inc., Ohio, USA). One microgram of RNA was reverse transcribed using an oligo-[dT]12–18 primer and the MMLV reverse transcriptase (Thermo Fisher Scientific Inc.-Life Technologies-Invitrogen, Massachusetts, USA). Produced cDNA was amplified by semi-quantitative (sq) PCR with a Bio-Rad T100 Thermocycler (Bio-Rad, California, USA), using chorionic gene-specific oligonucleotide primers (Table S1 ). PCR fragments were resolved in 2–3% agarose gels according to standard procedures. Data were collected from three independent experiments of different fly crosses per biological condition tested.
5
Confirmation of tccZ Gene Transformation
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Primers specific to the tccZ gene were designed manually using OligoAnalyzer Tool (Integrated DNA Technologies, idtdna.com ). The forward and reverse sequence were 5′-ATGCTTAAATCCGCGCTTTTTCTC-3′ and 5′-CTAATTACGCTTAACCCCATAGTC-3′, respectively. To confirm successful transformation, half a colony was mixed with 12.5 µl 2× Master Mix, 10.5 µl of nuclease-free water and 1 µl of each primer (10 µM) to make up a total of 25 µl reaction for PCR. Amplification of the tccZ gene was carried out in a Bio-Rad T100 Thermocycler (Bio-Rad, USA) using the following parameters: initial denaturation at 94 °C for 30 s, followed by 35 cycles at 94 °C for 30 s, 50 °C for 30 s and 68 °C for 1 min, and a final extension at 68 °C for 10 min. The PCR amplicons were confirmed by electrophoresis on 1 % agarose gel (90 V for 40 min) with 50 bp DNA ladder (New England Biolabs, USA). The other half of the colony was spread on LB agar supplemented with 50 µg ml−1 of kanamycin and incubated overnight at 37 °C (to be used for plasmid extraction).
6
Quantifying s38 Expression in Drosophila
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Control and s38-targeted female transgenic flies were dissected in Drosophila Ringer’s buffer and stage 14 follicles were hand-collected from isolated ovaries. Total RNA was extracted following a Trizol-based protocol (Molecular Research Center Inc., OH, USA), according to manufacturer’s instructions. One microgram of RNA was reverse transcribed, using an oligo-[dT]12–18 primer and the MMLV reverse transcriptase (Thermo Fisher Scientific Inc.-Life Technologies-Invitrogen, MA, USA). Produced cDNA was amplified by PCR with a Bio-Rad T100 Thermocycler (Bio-Rad, CA, USA), using s38 gene-specific oligonucleotide primers (Table S1 ). Actin served as gene of reference (control). PCR fragments were resolved in 2% agarose gels, according to standard procedures. RT-PCR assays were repeated three times, using independent fly crosses.
7
DNA Extraction and 16S rRNA Amplification
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For DNA extraction from isolates, a pellet was formed with low-speed centrifuging (7,000 × g) of isolates that had been grown on R2A at 4°C for 4 weeks, and DNA was extracted with a Qiagen DNA Power Soil Kit (Qiagen, Germany). To amplify the 16S rRNA gene of the isolates, a PCR Master Mix was prepared as follows: 0.25 μl of Speedstar Taq polymerase (TaKaRa Bio, United States), 4 μl of 2.5 mM deoxyribonucleotide triphosphates (dNTPs), 5 μl of 10 × Fast Buffer 1 (TaKaRa Bio, United States), 10 μl of 27F primer (5′-AGAGTTTGATYMTGGCTCAG-3′) 10 μl of 1492R primers (5′-TACGGYTACCTTGTTACACTT-3′) (Frank et al., 2008 (link)) (Eurofins Genomics, United States), 29.25 μl of dH2O, and 2 μl of DNA, totaling 200 μl of volume per sample. Samples underwent PCR thermocycling in a BioRad T100 ThermoCycler (BioRad, United States) for 95°C for 1 min, 95°C for 5 s, and 65°C for 20 s. The last two steps were repeated 34 times. After the thermocycler, samples were dyed with 6 × TriTrack DNA Loading Dye (Thermo Fisher Scientific, United States). Visualization of the PCR product was compared to a GeneRuler 1 kb Plus DNA Ladder (Thermo Fisher Scientific, United States). The samples were then placed into a 1.5% agarose gel with Midori green DNA stain at 90 V for 45 min with BioRad PowerPac Basic (BioRad, United States) for PCR product verification.
8
Kapa Hyper Prep Kit Protocol for Targeted Sequencing
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1 µg of genomic DNA per sample was processed using the Kapa Hyper Prep Kit following manufacturer’s recommended protocol (Kapa Biosystems, Inc., Wilmington, MA, USA). Briefly, samples were enzymatically sheared, A-tailed, and ligated to standard Illumina dual indexed adapters. Libraries were purified using AMPure XP beads (Beckman Coulter, Brea, CA, USA) and amplified in a Bio-Rad T100 Thermocycler (Bio-Rad, Hercules, CA, USA). Libraries were again purified using AMPure XP beads and validated with the TapeStation D1000 ScreenTape (Agilent Technologies, Santa Clara, CA, USA). Libraries were then hybridized to biotinylated target probes for the DBA following manufacturer’s recommended protocol (IDT). After hybridization, the captured DNA was captured with magnetic streptavidin beads and purified with several wash buffers in order of decreasing stringency. Libraries were again briefly amplified and purified using AMPure XP beads before final validation on with the TapeStation D1000 ScreenTape (Agilent Technologies). Up to 96 samples were normalized and pooled together for sequencing. Sequencing was conducted on the Illumina NextSeq 500 using 150-bp paired-end reads (Illumina Inc, San Diego, CA, USA).
9
Genotyping of Milk Protein and Lipid Genes
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Genotyping was accomplished using PCR-based methods. In particular, a duplex Artificially Created Restriction Site (ACRS)-PCR described by Pauciullo et al. [22 (link)] was performed for the AJ005430: c.578C>T at the CSN1S1 (αs1-CN) and HQ677596: c.536C>T at the CSN3 (k-CN). Additionally, Restriction Fragment Length Polymorphism (RFLP)-PCR, as described by Gu et al. [28 (link)] and Gu et al. [27 (link)], was used for genotyping the FM876222: g.133A>C at SCD (Stearoyl-CoA desaturase) and the AWWX01438720.1: g14229A>G at LPL (Lipoprotein lipase), respectively. PCR amplification was carried out using BioRad T100 thermocycler (BioRad, Hercules, CA, USA). The digestion products were analyzed directly via electrophoresis on a 2.5% agarose gel in 0.5× TBE buffer and stained with SYBR green nucleic acid stain (Lonza Rockland Inc., Rockland, ME, USA) (Figure S1 ).
10
PCR Amplification and Gel Electrophoresis
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The cDNAs were amplified by PCR with GoTaq® DNA Polymerase (Promega, Madison WI, USA) according to manufacturer’s instructions. The reactions were carried out with a Biorad T100 thermocycler (BioRad, Hercules CA, USA) with the following amplification conditions: 3 min at 95 °C, 50 cycles of: 30 s at 94 °C, 20 s at 51 °C and 30 s at 72 °C; and a final step at 72 °C for 5 min. The PCR products were loaded on a non-denaturing 12% polyacrylamide gel and migrated in 1X TBE buffer for 15 min at 50 V and then 45 min at 100 V. The gels were revealed after a 10 min incubation in a TBE-SYBR Gold Nucleic Acid Gel Stain 1X solution (Molecular probes, Invitrogen). The image captures of the gels were performed under a UV camera.
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