Mgcl2

RNA was extracted from E14.5 freshly dissociated and purified ENCCs, and from adult whole brain as described above. The concentration of total RNA in each sample was measured using a NanoDrop ND-1000 spectrophotometer. cDNA was synthesised using the iScript Advanced cDNA Synthesis Kit for RT-qPCR (Bio-Rad); 100-350ng of total RNA was used in a final reaction volume of 20 μl according to the manufacturer’s instructions. Control reactions using no reverse transcriptase or substituting cDNA with water were run in parallel for each tissue.
RT-PCR was conducted using intron-spanning specific primer pairs (S1 Table) and a touchdown PCR (TD-PCR) cycling program (S2 Table). A standard RT-PCR protocol was used consisting of MangoTaq (0.2 μl, Bioline), dNTP Mix (0.8 mM, Bioline), MgCl₂ (2 mM, Bioline), and primer pairs (1 μM each) in a final reaction volume of 20 μl. RT-PCR products (5–15 μl) were resolved by gel electrophoresis on a 1–2% agarose gel, containing either ethidium bromide (0.06 μg, Sigma) or GelRed (0.5x, Biotium), together with 100 bp DNA Ladder (Life Technologies) or Ready-to-Use 100 bp DNA Ladder (Biotium) to estimate product size. Control reactions were run in parallel for each RT-PCR reaction.

RAPD-PCR reactions were performed using the OLP 13 primer (5’-ACCGCCTGCT-3’) (Zare et al., 2019 (link)), and a 25μl reaction mix, containing 3mM MgCl2 (Bio Line, London, UK), 0.5 mM dNTPs (New England BioLabs, UK), 1 µM of primer, 1 U of Taq DNA polymerase (Bio Taq DNA polymerase, Meridian Bioscience, USA) in 1X PCR buffer (Meridian Bioscience, USA) and 3 μl of template dsDNA, was prepared. PCR was performed as follows: 1 cycle at 94°C for 5 min, followed by 40 cycles at 93°C for 1 min, 37°C for 1.5 min, and 72°C for 1 min, with a final extension at 72°C for 7 min (Mastercycler pro, Eppendorf, Germany). Amplification products were solved by electrophoresis on 1% agarose-TBE gel, stained with 5µl of “Midori Green Advance DNA Stain” (Nippon Genetics Europe GmbH, Germany), visualized under UV transilluminator (UVP Inc., Cambridge) and photographed with DigiDoc-It (UVP, Cambridge) system. To guarantee the reproducibility of RAPD-PCR profiles, the reactions were carried out in triplicate.

Genomic DNA was extracted from frozen seedling leaves following Murray and Thompson (1980) (link), as modified by Hernández et al. (2001) (link). The absence of Ph1 was verified using a PCR assay described by Wang et al. (2002) (link). Each 30 μL PCR contained 1x PCR buffer with MgCl₂ (Bioline USA, Taunton, MA, USA), 0.25 mM dNTP, 0.17 μM primers, 0.02 U/μL Taq DNA polymerase (Bioline USA), and 20 ng template. The reaction was first denatured (94^∘C/5 min), and then subjected to 35 cycles of 94^∘C/60 s, 51^∘C/60 s, and 72^∘C/60 s, followed by a final extension (72^∘C/7 min). The PCR products were electrophoretically separated through a 1% agarose gel and visualized by EtBr staining. The presence of each Hordeum sp. chromosome was based on PCR assays described by Liu et al. (1996) (link) and Hagras et al. (2005) (link) as detailed in Table 2. The composition of these PCR reactions was as above, while the amplification regime comprised an initial denaturing step (94^∘C/5 min), followed by 35 cycles of 94^∘C/15 s, 50–65^∘C (primer dependent, see Table 2) /30 s, 72^∘C/60 s, and completed by a final extension (72^∘C/6 min). The amplicons were separated as described above.