Iptg x gal

All the DNA of CRC tissues was subjected to bidirectional Sanger sequencing, the sequences of the primers as follows: forward (5′-ACAGTTCATTACGATACACG-3′) and reverse (5′-CCCAAGGTACATTTCAGATAAC-3′). All the mutation-negative amplicons identified by direct sequencing were subjected to T-A cloning. The 450bp amplicon was separated with 1.5% agarose electrophoresis and purified through the Qiaquick gel purification kit (Qiagen) before being combined with pMD19-T simple vector master mix (Takara BIO). 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 onto the IPTG/x-GAL (Invitrogen) coated ampicillin-LB agar dishes according to the instructions of the manufacturer. After 37°C incubation for 16hours, the white clones were picked up and again enriched in the ampicillin-LB broth at 37°C overnight. The monoclonal colonies collected were extracted with Plasmid Mini Kit (Qiagen) according to the manufacturer's instruction. All the Sanger sequencing assays were performed on an Applied Biosystems PRISM 3130 genetic analyzer in the Invitrogen Laboratory of Technical Services (Shanghai, China).

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.