Jumpstart taq polymerase

Genotyping of F0 and F1 animals was performed by harvesting fin clips from adult mosaic females as well as F1 animals under anesthesia (0.1% phenoxyethanol) and F1 eggs (1–2 cell stage) from mosaic females crossed with vasa:eGFP males. These samples were lysed with 5% chelex containing 100 μg of proteinase K at 55 °C for 2 h and then 99 °C for 10 min. The extracted DNA was subjected to PCR using the AccuPrime system (Promega) for slc29a1a, Advantage 2 system for nucleoplasmin 2b (npm2b), and Jumpstart Taq polymerase (Sigma-Aldrich) for otulina and vasa:eGFP. The primers are listed in Additional file 2.

Genomic DNA was extracted from earthworm tissues using the Qiagen DNeasy Blood and Tissue Kit (QIAGEN, Valencia, CA, USA). Regions of eight molecular markers, including three nuclear rRNAs (18S, 5.8S and 28S), two mitochondrial rRNAs (12S and 16S), and three mitochondrial protein coding genes (cytochrome c oxidase subunit 1 and 2 (COI, COII) and NADH dehydrogenase subunit 1 (ND1)), were acquired using polymerase chain reaction (PCR) with primers listed in S2 Table. PCR was conducted in a 50 ul total volume with 1x reaction buffer, 1.25 units JumpStart Taq Polymerase (Sigma, St Louis, MO, USA), 200 uM of each dNTP, 1.5 mM MgCl₂, 0.24 mg/mL BSA, 5% DMSO, 200 nM of each primer, and 10 or 20 ng template DNA. Cycling conditions were set to one cycle of 94°C for 2 min, followed by 35 cycles of 94°C for 15 s, 45°C (for COI), 47°C (for COII and ND1), 49°C (for 16S rRNA) or 50°C (for 12S, 18S, 5.8S and 28S rRNAs) for 15 s, and 72°C for 90 s, with a final cycle of 72°C for 5 min. The amplified products were sequenced at Beckman Coulter Genomics using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster, CA, USA) and analyzed on an ABI PRISM 3730XL (Applied Biosystems). Chromatograms were visualized and assembled in DNA Baser v4.31.0 (Heracle BioSoft, Romania). All new sequences have been deposited in GenBank under the accession numbers KX651115-KX651415.

Samples of wound tissue were homogenized in Tri-Reagent using a Tissumizer rotor-stator homogenizer (Teledyne Tekmar, OH). RNA was isolated by either chloroform phase separation with an Ambion Ribopure kit (Life Technologies, NY), or directly from Tri-Reagent with a Direct-Zol miniprep plus kit (Zymo Research, CA), and subsequently reverse transcribed into cDNA using iScript supermix (Bio-Rad, CA). Genes of interest were then assayed by qPCR using mouse TaqMan probes for STAT4 (Mm00448890_m1) Ccl2 (Mm00441242_m1) and Ccr2 (Mm00438270_m1) (Life Technologies, NY) with JumpStart Taq polymerase (Sigma-Aldrich, MO), 3 mM MgCl₂, and 200 μ M dNTPs (Promega, WI) using a Bio-Rad CFX96 C1000 thermocycler. A standard TaqMan cycling protocol was performed, consisting of a 10 min 95°C hold, followed by 40 rounds of 15 sec at 95°C and 1 min at 60°C. 18s expression was used as a housekeeping control for data normalization.

RNA was isolated using TRIzol (Life Technologies, Grand Island, NY, USA) and converted to cDNA using the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA). The concentration of RNA was measured using a spectrophotometer (Nanodrop 2000, Thermo Fisher Scientific). Gene expression was assessed using Jumpstart Taq Polymerase (Sigma-Aldrich) and SYBR Green nucleic acid stain (Life Technologies). Threshold crossing values for each gene were normalized to glyceraldehyde phosphate dehydrogenase (GAPDH) gene expression. mRNA expression was then normalized to fold change relative to controls.