Mi plasmid miniprep kit

Large fragments of TB40-BAC4 were cloned in plasmid pBR322 by using the gap repair method described previously [30 (link)] with a few modifications. The low-copy plasmid pBR322 was first linearized by digestion with HindIII. The replicative sequences and the amp^R marker were PCR-amplified using primers containing approx. 20 nucleotides of homology to pBR322 and 40 nucleotides of homology to the TB40-BAC4 fragment to be subcloned. Unique restriction enzyme sites (SmiI and MssI) were also introduced through the primers. The PCR product was then purified by gel electrophoresis and gel purification. Purified vector DNA (150 ng) was used to transform recombination-competent GS1783 containing TB40-BAC4 with a previously kan^R/I-SceI-tagged region. Bacterial clones carrying the recombinant plasmid were obtained by plating on LB agar plates containing kanamycin and ampicillin. DNA of positive recombinants was isolated using mi-Plasmid Miniprep Kit (Metabion, Planegg, Germany) and examined by digestion with SmiI+MssI, HindIII, and BamHI.

To validate the candidate mutations detected by DNA sequencing, we selected several mutated positions with ≥20% variant calls (Supplementary Table S2). After PCR, amplicons were analyzed by Sanger sequencing (ABI3130 Genetic Analyzer; Applied Biosystems, Life Technologies). Primer sequences are available from the authors upon request. For mutations with <20% of variant calls, amplicons were cloned. PCR products were ligated into the pGEM T-easy vector (Promega, Madison, WI, USA). After transformation in XL1-Blue (Stratagene, Waldbronn, Germany) JM109 (Promega) competent cells, plasmids were isolated (mi-Plasmid Miniprep Kit, Metabion, Planegg/Steinkirchen, Germany) and individual clones were sequenced by Sanger sequencing. Sequences of PCR products were compared to the corresponding germ line sequences with SeqScape v2.5 software (Applied Biosystems).

To generate pMKPccdB vector with BsaI, BsmBI or AarI cloning/restriction sites, Pol-I/ccdB/Pol-II cassettes containing the three different cloning/restriction sites were transferred from the respective pMPccdB vector versions [39 (link)] using “FastDigest” BsrBI enzyme (Thermo Scientific, USA) at 37°C for 15 min. The gel-purified cassettes were ligated into the EcoRV-blunt ends of pSMART-LC-Kan vector using T4 DNA ligase (Thermo Scientific, USA) according to the manufacture instructions. The ligation reactions were then transformed into “One Shot ccdB Survival 2 T1R” competent E. coli cells (Invitrogen, USA) according to the manufacture instructions and cultured on LB agar plates. 16–18 h later, five individual colonies were picked up and cultured for 18 h at 37°C in 5 ml of LB media containing 30 μg/ml of Kanamycin (SERVA, Germany) followed by plasmid DNA extraction using mi-Plasmid Miniprep Kit (Metabion, Germany). The bacterial culture containing the correct plasmid DNA construct, confirmed by enzymatic digestion and sequencing, was subjected to large scale culture (250 ml LB media) and plasmid DNA extraction/purification using NucleoBond Xtra Maxi (Macherry-Nagel, Germany).