Plasmid miniprep dna purification kit

All DNA manipulations, polymerase chain reactions (PCRs), restriction digests, ligations, and DNA electrophoresis, were performed as previously described (Sambrook and Russell, 2001 ). Plasmid and genomic DNA were isolated using a Plasmid Miniprep DNA purification Kit and Bacteria & Yeast Genomic DNA Purification Kit (EurX), respectively. When the amplified fragments were used for cloning, PCR was performed using DreamTaq DNA polymerase or Phusion High-Fidelity DNA polymerase (Thermo Scientific). Oligonucleotide primers for PCR and sequencing were purchased from Sigma Aldrich and are listed in Table S2. DNA fragments amplified by PCR were purified using a PCR/DNA Clean Up kit (EurX). The plasmids used in this study are described in Table S1. DNA sequencing was performed by Genomed S.A. (Warsaw, Poland).

In order to confirm the integration of the transgene into the hairy roots genome, total genomic DNA from 7 lines (SOPSS1-7) showing the best growth rate were isolated using Genomic Mini AX Plant kit (A&A Biotechnology) following the manufacturer’s procedures. Additionally, plasmid DNA (isolated using Plasmid Miniprep DNA purification kit, EURx, Gdansk, Poland) was also isolated from A. rhizogenes cells as a positive control. PgSS1 sequence specific primers (For-5′-gcatggaagagctttgacaac-3′, Rev-5′-atgggaagtttgggggcaat-3′). Amplification conditions: three minutes at 95 °C; 35 cycles of 30 s at 95 °C, 30 s at 52 °C, one minute at 72 °C; finally, five minutes extension at 72 °C. Final volume of all samples was 25 μL (1 μL of each primer (10 μM), 0.5 μL of DNA (50 ng/μL), 12.5 μL of PCR Mix Plus master mix (A&A Biotechnology, Gdynia, Poland) and 11 μL of PCR Ultra-Pure Water H2O (Blirt). DNA amplification was performed in a Biometra UNO II thermal cycler. The PCR products were then visualized on 1.5% agarose gel stained with ethidium bromide.

Genomic and plasmid DNA isolations were performed with Bacterial & Yeast Genomic DNA Purification Kit (EURx Sp. z o.o., Gdańsk, Poland) and Plasmid Miniprep DNA Purification Kit (EURx Sp. z o.o., Gdańsk, Poland), respectively, according to the manufacturer’s protocols. Molecular cloning and transformation were performed according to standard protocols [70 ]. FastDigest restriction endonucleases were purchased in Thermo Fisher Scientific (Waltham, MA, USA). PCR was performed with high-fidelity Platinum SuperFi II DNA Polymerase (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s recommendations. The plasmids constructed and used in this work are listed in Supplementary Tables S2, S4, and S6–S8. The primers used in this work were synthesized at Genomed S. A. (Warsaw, Poland) and are listed in Supplementary Tables S1, S2, and S5–S7. Sanger DNA sequencing of plasmid constructs prepared in this work was performed in Genomed S. A. (Warsaw, Poland).