Accustart 2 geltrack supermix

Genomic DNA was extracted using MasterPure Complete DNA and RNA Purification Kits (Epicentre). PCR screening was performed using AccuStart II GelTrack SuperMix (QuantaBio) with annealing temperature (T_a) at 64 °C. The product sizes are 1542 bp for H1 parental and 2097 bp for H1_SOD1-SNAP. Sample for Sanger sequencing (Eton Bioscience) was prepared using Q5 Hot Start High-Fidelity DNA Polymerase (NEB) with the same primer set at T_a 66 °C and extension for 1 min each cycle.

Genomic DNA was extracted using the MasterPure Complete DNA and RNA Purification Kit (Epicentre). PCR for screening was performed using AccuStart II GelTrack SuperMix (QuantaBio) with the annealing temperature (T_a) of 60 °C. Genomic fragments for Sanger sequencing were amplified using Q5 Hot Start High-Fidelity DNA Polymerase (NEB) with the same primer set at T_a = 66 °C.

DNA was extracted from cloacal swab and stool samples using the Powersoil Pro DNA Extraction kit (Qiagen) according to manufacturer instructions. Samples were screened for the presence of parabasalid protists by amplifying DNA using custom pan-parabasalid primers, which we designed to bind to regions of the internally transcribed spacer (ITS) that are highly conserved among the parabasalid lineage (panParabasalid-F 5’-CCACGGGTAGCAGCA-3’, panParabasalid-R 5’-GGCAGGGACGTATTCAA-3’). These primers amplify an approximately 1.1kb ITS amplicon. DNA was amplified using the Accustart II Geltrack Supermix (Quantabio) using touchdown PCR, with a starting annealing temperature of 72°C dropping to 65°C over 15 cycles, followed by 35 cycles with an annealing temperature of 65°C. A 1-minute extension at 72°C was used for both stages. The presence of parabasalid DNA in the samples was initially assessed by agarose gel confirmation, followed by verification and species identification using Sanger Sequencing (Molecular Cloning Lab). Phylogenetic trees were created using Phylogeny.fr^{23 (link)} based on ITS sequences, using default parameters.