Phire plant direct pcr master mix

Detection of the PKS-I gene was carried out by amplification of DNA Template as much as 1 l with primers MDPQQR f (5′-RTRGAYCCNCAGCAICG-3′) and HGTGT r (5′-VGTNCCNGTGCCRTG-3′) (El Samak et al., 2018), with concentrations 10 mM, 0.5 µL each, 7.5 µL Thermo Scientific2X Phire Plant Direct PCR Master Mix, and 6 µL ddH2O, and 0.5 µL DNA template from each actinomycetes isolate. The amplification process was carried out in 40 cycles, as follows: initial denaturation stage (95 °C, 5 min), followed by 10 cycles of denaturation stages (95 °C, 1 min), annealing (60 °C, 30 sec), extension stage (72, 1 min), then 30 cycles of denaturation (95 °C, 1 min), annealing (40 °C, 30 sec), extension stage (72 °C, 1 min), and final extension (72 °C, 10 min).
PKS-II gene amplification was carried out by mixing primer pairs PF6 (5′-TSGCSTGCTTGGAYGCSATC-3′) and PR6 (5′TGGAANCCGCCGAABCCGCT-3) (El Samak et al., 2018) with a concentration of 10 mM, 0.5 µL each, 0.5 µL of extracted DNA template, 7.5 µL of Thermo Scientific2X Phire Plant Direct PCR Master Mix, and 6 µL of ddH₂O. PCR amplification was carried out in 30 cycles, as follows: initial denaturation (96 °C, 5 min), followed by denaturation (96 °C, 1 sec), annealing (58 °C, 1 min), extension (72 °C, 1.5 min), and the final extension (72 °C, 10 min).

PCR amplification of genomic Chlamydomonas DNA was performed in a 96-well format using Phire Plant Direct PCR Master Mix (Thermo Fisher Scientific; dilution buffer protocol) and crude cell extracts. Cells were grown in 180 µl of TAP(-Arg) medium in 96-well plates until all wells had turned uniformly green in color. A 40-µl aliquot of each cell culture was then transferred to a 96-well V-bottom culture plate and centrifuged at 2000 g for 10 min at RT. The supernatant was removed, and the pellet was thoroughly resuspended in 20 µl of dilution buffer, incubated for 5 min at RT, centrifuged again at 4000 g for 10 min at RT, and used for PCR. PCR was performed according to the manufacturer’s instructions and according to the protocols of Greiner et al. (2017) (link). Oligonucleotide sequences used for screening are listed in Supplementary Table S4.

Populus alba x tremula clone 717-1B4 was used for stable transformation as described (Leple et al., 1992 (link)). Transformed shoots were selected by regenerating on media containing hygromycin. The rooted plants were propagated and used for further genotyping. Two rounds Hi-Tom PCR were preceded to obtain amplicons using Phire Plant Direct PCR Master Mix (ThermoFisher).