Pcr2.1 topo ta cloning vector

Degenerated primers used to amplify internal cDNA fragments of Senegalese sole ssvkorc1 and ssvkorc1l1 were designed in regions conserved among fish vkor sequences retrieved from GenBank and SoleaDB databases using on-site Basic Local Alignment Search Tool (BLAST) facilities. cDNA fragments of ssvkorc1 and ssvkorc1l1 were used to design gene-specific primers to amplify cDNA ends by RACE-PCR following the manufacturer instructions, and later the full-length cDNAs. PCR fragments were cloned in pCR2.1-TOPO TA cloning vector (Invitrogen, Alcobendas, Spain) and sequenced using CCMAR sequencing facilities. Gene-specific primers for the quantification of ssvkorc1 and ssvkorc1l1 relative gene expression by qPCR were designed. The sequences of the primers used in this work are presented in Table 1.

Total RNA was isolated from the leaves of 4-week-old O. sativa seedlings, and the cDNA was synthesized by reverse transcription-polymerase chain reaction (RT-PCR). The OsMDHAR coding region was amplified from the cDNA by PCR using Taq and Pfu polymerases (Roche, Basel, Switzerland). The reaction conditions were as follows: initial denaturation at 94°C for 3 min, followed by 30 cycles of 94°C for 30 s, 56°C for 30 s, and 72°C for 2 min, and a final extension at 72°C for 7 min. The primer set used for PCR cloning of the OsMDHAR gene is shown in S1 Table. The PCR product was ligated into the pCR2.1^® TOPO-TA cloning vector (Invitrogen, Carlsbad, CA, USA). After sequence confirmation and digestion with EcoRI, the DNA fragment containing the OsMDHAR gene was ligated into the yeast expression vector p426GPD (Euroscarf, Frankfurt, Germany). The construct was transformed into S. cerevisiae BY4741 cells (S2 Table) and derivatives using the PEG/LiCl method [24 (link)]. Transformants were selected by plating cells on minimal agar medium (0.67% yeast nitrogen base without amino acids and with ammonium sulfate and 0.192% yeast synthetic drop-out medium lacking uracil, with 2% glucose, and 2% bacto agar) at 28°C for 3 days. Single colonies were restreaked, cultured under the same conditions, and then used for subsequent experiments.

Different alleles were cloned in pCR2.1 TOPO TA cloning vector (Invitrogen—Life Technologies) and plasmids were extracted with QIAprep Spin Miniprep Kit (Qiagen, Courtaboeuf, France), according to the manufacturer's protocol. Amplifications using M13R and M13F primers were performed with EconoTaq after an initial denaturation step of 95°C for 5 min, followed by 40 cycles of 95°C for 10s, 55°C for 20s and 72°c for 20s and a final elongation of 72°C for 7 min. ABI 3730XL sequencer was used by Genoscreen (Lille, France) to sequence alleles.

Stocks of antibiotics and heavy metals were prepared with sterile distilled water (dH₂O) at 10⁴ µg·mL⁻¹. Antimicrobial agents were freshly diluted in dH₂O to appropriate concentrations as necessary. Selection for the transfer of pCR2.1^®-TOPO^® TA cloning vector (Invitrogen^TM, Life Technologies, Carlsbad, CA, USA) and the broad-host range plasmid, pBBR1MCS-5, was achieved on NA containing ampicillin (100 µg·mL⁻¹) and gentamicin sulfate (10 µg·mL⁻¹), respectively. A nalidixic acid resistant (7 µg·mL⁻¹) laboratory strain E. coli DH5α was used for construction of the lead biosensor construct. Heavy metals used for testing of the biosensors included: sodium arsenite (As(III)), cadmium chloride (Cd(II)), copper chloride (Cu(II)), chromium oxide (Cr(VI)), lead nitrate (Pb(II)), mercury chloride (Hg(II)) and zinc chloride (Zn(II)). Antibiotics (ampicillin, gentamicin sulphate and nalidixic acid) were stored at 4 °C and heavy metal compounds were stored at room temperature. All antimicrobial agents were obtained from Sigma-Aldrich Pty Ltd. (St. Louis, MO, USA).

RT-PCR or PCR products were gel-purified from 1X TAE gel (QIAquick gel extraction Kit, Qiagen), and were cloned into pCR2.1-TOPO TA-cloning vector (Invitrogen) and subsequently sequenced. The vector contains an IPTG-inducible Plac promoter to drive expression of the insert. It also includes a single NcoI site that was used for linearizing the plasmid, and two EcoRI sites used for plasmid digestion (see Figure 7A).
Wild-type (WT) construct was made with primer pair BcISt1c left/right for amplification before cloning. The deletion constructs Δ5′DR, Δ3′DR and ΔORFB were amplified by inverse PCR with outward primers (see Supplementary Table S2) from the WT plasmid construct as template using Pfu Turbo in order to remove specific IStron regions, and then ligated with T4 ligase (New England Biolabs). The various constructs were transformed into either E. coli xl-1 (recA+) or E. coli SCS110 (recA−) strains.