Pureplasmid mini kit

The C. striatum isolates previously identified using mass spectrometry were further identified by 16S rRNA gene amplification and sequencing. High-fidelity PCR amplification was performed using the universal primers of the 16S rRNA gene of the C. striatum strain (Table S3) [27 (link)], and no template was added as a negative control. The PCR products were electrophoresed and purified (Gel Extraction Kit, omega, Norcross, GA, USA), subcloned into the blunt-end vector (pEASY^®-Blunt Zero Cloning Kit, TransGen, Beijing, China), transformed into Escherichia coli DH5α competent cells, and spread on Luria-Bertani agar (ampicillin 0.1 mg/mL). PCR was performed using the colony as template, and the positive colonies were inoculated into the broth containing ampicillin overnight (37 °C, 200 rpm). Plasmids carrying the 16S rRNA gene fragment were extracted with PurePlasmid Mini kit (CWBIO) and sent for sequencing (AuGCT, Wuhan, China). The amplification and identification of the site-specific recombination element, the integrase gene, was also carried out according to similar steps. Primers of the integrase gene (Table S3) were designed with reference to the complete genome of the Corynebacterium striatum strain 216 (Accession: NZ_CP024932.1, 1618198-1619379).

Outer primers were used to amplify target DNA, and the amplified PCR products were purified with a gel extraction kit (Takara, Dalian, China), cloned into the pMD18-T vector (Takara, Dalian, China) and transformed into E. coli DH5α competent cells. Clones were sequenced by BGI Tech (BGI Tech, Shenzhen, China), and analyzed results. High fidelity clone was cultured and extracted plasmid with Pureplasmid Mini Kit (CWBIO, Beijing, China). Meanwhile, 1.0 × 10⁷ copies/μL–1.0 × 10² copies/μL substances were used for constructing standard curve.

To indirectly access the X-chromosome inactivation patterns of two female patients in family X3, the cDNAs of cultured skin fibroblasts were amplified for 25 cycles and inserted into pGEM-T Easy plasmid. Then, the vector plasmids with cloned insert were transformed into DH5α competent E. coli cells and multiplied in the Luria-Bertani (LB) broth and spread evenly on the IPTG/x-GAL (Invitrogen, United States) coated ampicillin-LB agar plates for 16 h at 37°C. Forty-eight collected monoclonal colonies were picked up and extracted using PurePlasmid Mini Kit (Cwbio, China). The plasmid DNA was sequenced using T7/SP6 primers. Sequence analysis and alignment were performed using Chromas 2.31 and Vector NTI Advance 10.