Qiaprep spin miniprep kit 250

A DNA fragment encoding the antigenic TTFC polypeptide was polymerase chain reaction-amplified from pTETtac::ttfC [26 (link)] using two pairs of primers: F-ttfC-N/R-ttfC-N and F-ttfC-C/R-ttfC-C (Table 1). Similarly, the entire open reading frame of flaB from V. vulnificus CMCP6 was amplified from pCMM250 [9 (link)] using two primer pairs: F-ttfC-N/R-ttfC-N and F-ttfC-C/R-ttfC-C (Table 1).
Amplified DNA fragments were initially cloned into the pCR2.1 TOPO vector (Invitrogen, Inc., Carlsbad, CA, USA), yielding plasmids pCMM8209, pCMM8210, pCMM8211, and pCMM8212. DNA fragments were excised using appropriate restriction enzymes and isolated from agarose gels using the QIAEX II gel extraction kit (Qiagen, Hilden, Germany). Plasmid DNA was purified using a QIAprep Spin Miniprep Kit 250 (Qiagen). The 1.3 kb ttfC and 1.1 kb flaB fragments were then cloned into the pTYB12 vector (New England BioLabs, Beverly, MA, USA), yielding plasmids pCMM8213, pCMM8214, pCMM8215, and pCMM8216 (Table 2, Fig. 1A). DNA sequences of the resulting expression vectors were confirmed by the dideoxy-chain termination method. Structure prediction was performed as previously described [28 (link)].

The target sequences were amplified from synthetic DNA using oligonucleotides (Supplementary Table S1) from Microsynth (Balgach) and cloned into the NotI and XhoI restriction sites of psiCHECK-2 plasmid (C8021; Promega, Fitchburg). The primers used for cloning as well as the inserted sequences in the psiCHECK2 vector are reported in the supplementary material. The ligated fragment was transformed into Subcloning Efficiency™ DH5α™ Competent E. coli (18265-017; Life Technologies, Carlsbad), plated on LB agar with 100 μg/ml Amp and incubated for at least 16 h at 37°C. The single colonies were picked and resuspended in water. Clones were screened by a Colony polymerase chain reaction (PCR) for the desired plasmid. Positive clones were used to make glycerol stocks and DNA minipreps were sequenced by GATC (Cologne). All the PCR purifications were performed following the protocol of MinElute^® PCR Purification Kit (250) (28006; Qiagen, Venlo). Minipreps were performed following the protocol of QIAprep^® Spin Miniprep Kit (250) (27106; Qiagen, Venlo). Digestions were performed following the guideline of the NEB website (http://www.neb.com) using NotI and XhoI according to the supplied protocol (NEB, Ipswich). The ligations were performed with ratios of 1:3, 1:4 (vector to insert) using the dephosphorylated psiCHECK-2 vector with T4 DNA Ligase (M1804; Promega, Fitchburg).