Taq polymerase

Total RNA was extracted from endometrial, conceptus, and chorioallantoic tissues using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s recommendations. The quantity of RNA was assessed spectrophotometrically, and the integrity of RNA was validated following electrophoresis in 1% agarose gel. Four micrograms of total RNA were treated with DNase I (Promega, Madison, WI, USA) and reverse transcribed using SuperScript II Reverse Transcriptase (Invitrogen, USA) to obtain cDNA. The cDNA templates were then diluted 1:4 with sterile water and amplified by PCR using Taq polymerase (Takara Bio, Shiga, Japan). The final PCR reaction volume of 50 μL included 3 μL of cDNA, 5 μL of 10× PCR buffer, 4 μL of dNTP mix (2.5 mM), 1 μL of each primer (20 μM), 0.3 μL of Taq polymerase (Takara Bio, Japan), and 36.7 μL of H₂O. Initial denaturation was performed at 94°C for 5 min; 40 cycles of amplification were carried out at 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s; and final extension was conducted at 72°C for 10 min. The PCR products were separated on 2% agarose gels and visualized by ethidium bromide staining. The identity of each amplified PCR product was verified by sequence analysis after being cloned into the pCRII vector (Invitrogen, USA).

Polymerase Chain Reaction – Restriction Fragment Length Polymorphism was employed to analyze the Single Nucleotide Polymorphism (SNP) LEP-R c.668A>G (p.Gln223Arg, rs1137101) of leptin receptor gene in the specimens. The reaction was performed in a final volume of 50 μl of reaction mixture which contained: 100 ng of genomic DNA, 5 μl PCR buffer (TaKaRa, Japan), 4 μl dNTP (10 mM, TaKaRa, Japan), 1 unit of Taq Polymerase (TaKaRa, Japan) and 0,5 μl of each primer (10 mM, Polgen, Lodz, Poland). Deionized H₂O was added. The amplification was completed in Thermal Cycler PTC-100 TM (MJ Research, INC, Waltham, MA, USA) in conditions as follows: initial denaturation in 94 °C (3 min) which was followed by 35 cycles of: denaturation in 94 °C (60 s), hybridization with starters in 65 °C (60 s) and finally augmented to 72 °C (90 s). Synthesis was concluded in 72 °C (7 min). Following starters were used:
forward: 5′-AAA CTC AAC GAC ACT CTC CTT-3′.
reverse: 5′-TGA ACT GAC ATT AGA GGT GAC-3′.
The PCF-RFLP product (20 μl of reaction mixture) was incubated overnight with 1 unit of restriction enzyme MspI (Fermantas) in 37 °C.

Genomic DNA was extracted from the muscle of the pereiopod of all 17 spiny lobsters. DNA was extracted using the AccuPrep^® Genomic DNA Extraction Kit (Bioneer, Daejeon, South Korea), following the manufacturer’s instructions. Concentration and purity of the extracted DNA were measured using a Thermo Scientific™ NanoDrop™ One microvolume UV-Vis spectrophotometer (Thermo Scientific, Wilmington, DE, USA).
A polymerase chain reaction (PCR) was performed to amplify the mitochondrial COI gene region using the HCO1490/LCO2198 universal primers, specially designed for invertebrates (Folmer et al., 1994 (link)). PCR was performed using a 50 µL reaction mixture, consisting of 100 ng genomic DNA, 0.25 µL Taq polymerase (Takara Bio Inc., Shiga, Japan), 5 µL 10X Ex. Taq DNA polymerase buffer (Takara Bio Inc.), 1 µL each of 10 µM forward and reverse primers, and 4 µL (2.5 mM) dNTPs (Takara Bio Inc.). The PCR thermal profile comprised an initial step of 5 min at 94 °C, followed by 30 cycles at 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 45 s, followed by a final extension at 72 °C for 5 min. The amplified PCR products were separated using 1% agarose gel electrophoresis, and target bands were purified using the AccuPrep^® PCR Purification Kit (Bioneer), according to the manufacturer’s instructions.

Mutations at sites 2063, 2064, and 2617 in the M. pneumoniae 23S rRNA gene domain V region were detected by direct sequencing of samples with a positive PCR result as described elsewhere (39 (link), 40 (link)). Briefly, by mixing primers (Sigma-Aldrich, China), Taq polymerase (TaKaRa Bio, Inc., Shiga, Japan), and extracted DNA, nested PCR was performed with a thermal cycler (TaKaRa Bio, Inc., Shiga, Japan). The purified PCR products were labeled with a BigDye Terminator version 3.1 cycle sequencing kit (Applied Biosystems, Foster City, CA, USA) and applied to an ABI Prism 3130xl genetic analyzer (Applied Biosystems, Foster City, CA, USA) in accordance with the manufacturer’s instructions. A sequence scanner (Applied Biosystems, Foster City, CA, USA) was used to determine gene mutations at each site.

Extensive manual curation of chloroplast and 45SnrDNA revealed different kinds of non-redundant sequence variations (SV) such as SNPs, InDels, and copy number variations. Inter-species and intra-species structural variations were analyzed for chloroplast and 45SnrDNA sequences from 28 Brassica and Raphanus genotypes (Tables S2–S5). Putative SNPs and InDels were manually analyzed using the file aligned with MEGA7. Tandem repeats were identified using the Tandem repeats finder (TRF) tool. To detect highly reliable variations, all predicted variations were manually curated for both chloroplast and 45SnrDNA. Some of the randomly selected and highly informative variations were validated by PCR analysis.
To validate the polymorphic regions of chloroplast and 45SnrDNA sequences, specific primers were developed for high-quality structural variations such as SNPs and InDels (Table S7). DNA templates from 28 genotypes were used for target analysis. Each PCR reaction contained 10 ng template DNA, 10 pM primers, 0.5 µM dNTPs, 2 units of Taq polymerase (TAKARA, Japan), and the final volume brought to 20 µl with sterile distilled water. The PCR reactions were 10 min at 95 °C; followed by 36 cycles of 30S at 94 °C, 30S at 55−62 °C, and 30S at 72 °C; with a final extension at 72 °C for 5 min. Amplified fragments were checked with 2% agarose gel electrophoresis to estimate the product size.