Faststart high fidelity pcr system

To design single-guide RNAs (sgRNAs), the online program CRISPRscan (www.crisprscan.org) was used (Moreno-Mateos et al., 2015 (link)). The gRNAs were designed to target exons, except for exon 1, to be as close as possible to the transcription start site and to have no predicted off-target effects. The sgRNAs were generated from annealed primers, one containing a minimal T7 RNA polymerase promoter, the target sequence, and a tail-primer target sequence and a generic tail-end primer (Vejnar et al., 2016 (link)). To generate primer dimers, the FastStart™ High Fidelity PCR System from Sigma-Aldrich was used. A solution was prepared, containing 1 mM forward sgRNA oligonucleotide, 1 mM reverse oligonucleotide consisting of 20-nucleotide overlap with sgRNA oligonucleotide and the Cas9-binding part, 0.8 mM dNTPS, 1× FastStart Buffer and 6.25 U/µl FastStart Taq polymerase in 20 µl total volume. Annealed DNA oligonucleotide dimers were generated by denaturation at 95°C for 5 min followed by annealing by reducing the temperature by 1°C per second over 20 s to 75°C and extension at 72°C for 10 min. The gRNAs were synthetized from annealed DNA oligonucleotides, containing a minimal T7 RNA polymerase promoter, with the mMESSAGE mMACHINE™ T7 ULTRA Transcription Kit (Invitrogen), according to the manufacturer's instructions.

The twelve different PCR primer pairs employed in this study were selected from the literature and from the BOLD primer dataset platform (http://www.boldsystems.org/index.php/Public_Primer_PrimerSearch) to target the four most commonly cited plant DNA barcoding loci (rbcL, matK, trnH-psbA, and ITS) (Table 1). Primers were validated according to the primer validation protocol described in the Fluidigm User Guide (Fluidigm PN 100–3770, San Francisco, California, USA), using the FastStart High Fidelity PCR System (Sigma-Aldrich). Amplicons were visualized using gel electrophoresis on an agarose gel (1.5% agarose, 90 V for 45 minutes). All primer pairs used in the validation protocol produced amplicons as anticipated and were retained for use on the Access Array.