Mango taq polymerase

For screening of putatively transformed shoots, leaf samples were collected from young green shoots grown in the SEM supplemented with kanamycin. DNA was extracted using a published crude extraction method [54 (link)]. A total reaction volume of 20 µL (× 1 PCR buffer, 0.2 mM dNTPs (Bioline), 0.2 µM of each primer (Sigma) (Additional file 1: Table S10), 2 mM MgCl₂ (Bioline), 1-unit Mango™ Taq polymerase (Bioline) and 40 ng of gDNA) was used in each PCR reaction. PCR products were separated by gel electrophoresis and visualised.
RNA was extracted from putatively transformed plants using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. RNA was treated with DNase (TURBO DNA-free kit Invitrogen) to remove any traces of genomic DNA. One µg RNA was reverse transcribed using iScript™ cDNA synthesis kits (Bio-Rad) according to the manufacturer’s instructions. A 1.5 µL aliquot from each cDNA sample was used in conventional PCR with gene-specific primers.

Seeds from Ox and fp2 (Pieper et al., 2016 (link)) were stratified for 1 week at 4°C and sown on 15.03.2016 on hydrated Jiffy plugs. Seedlings were first grown in a greenhouse without temperature or light control, and later transferred to the field on 13.04.2016. More details on experimental design can be found in Figure 5 and Figure 5—figure supplement 2. Genomic DNA was extracted from parent and progeny plants with Edwards Buffer and isopropanol precipitation and amplified with primers m458 (5’-GCCTAATCTTGCACAACACGAAATCT) and m459 (5’-GATTCTAAAGTTCTGTCAAAAGGAGAAACCTGA), designed with dCAPS Finder (http://helix.wustl.edu/dcaps/dcaps.html), to genotype SNP:2:2905982 by dCAPS. PCR was performed with Mango Taq polymerase (Bioline) under the following conditions: initial denaturation of 5 min at 95°C, 40 cycles of 30 s at 95°C, 30 s at 56°C and 30 s at 72°C, final extension of 10 min at 72°C. 1/5^th volume of the reaction was digested with 2.5 units of DdeI (New England Biolabs) for 2 hr at 37°C and migrated on a 3% agarose gel to resolve the uncut 141 bp amplicon for fp2, and the two cut fragments of 116 bp and 25 bp for Ox.

For amplification of the genomic region of interest, target site-specific PCR primers were designed using Primer Blast (NIH) and a touchdown PCR in combination with a Mango Taq Polymerase (Bioline, London, UK) was performed. The annealing temperature was adjusted according to the melting temperatures of the primers. TMEM43-specific point mutations were confirmed using following primers, eGFP_for: 5′-CATGGTCCTGCTGGAGTTCGTG-3′ and TMEM43-c.332_rev: 5′-TGGGATTTTCGCTGTGGTAGAA-3′, with an amplicon size of 797 bp; TMEM43-c.1073_for: 5′-TTCTACCACAGCGAAAATCCCA-3′ and TMEM43-c.1073_rev: 5′-CGTGTCCGAGCAACAAGGATGG-3′, with an amplicon size of 506 bp. The following primers were used to study tmem43 CRISPR mutations, tmem43-CRISPR_for: 5′-TGTGATTAGCGCGGGTTTCT-3′ and tmem43-CRISPR_rev: 5′-TCGCTGTACTCCACCCATTG-3′, with an amplicon size of 791 bp. PCR products were cleaned up using the ExoSAP-IT PCR Product Cleanup kit (Thermo Fisher Scientific) and sequenced by Microsynth Seqlab (Balgach, Switzerland) or Eurofins Genomics (Luxembourg). Sequencing traces were visualized and analyzed with SnapGene (San Diego, CA, USA).