Abi prism biodye terminator

Purified BSP amplification products were ligated to the pGEM-T Easy Vector and transformed into competent JM109 cells by using the pGEM-T Easy Vector system I (Promega, Madison, WI, USA). White colonies were selected from the ampicillin/X-Gal/IPTG plates and colony PCR was performed using vector-directed primers to confirm the presence of inserts based on their expected fragment size. Ten positive colonies from each plate were inoculated into LB medium with ampicillin for plasmid isolation. Plasmids containing the target DNA were extracted and purified by using the Plasmid Midi Kit (QIAGEN) and sequenced by using the ABI Prism BioDye Terminator version 3.1 sequencing system. Cytosine methylation results were analyzed using the CyMATE online methylation sequence analysis software48 (link). Average methylation level expressed as percentage (%) per site for each of the three types of cytosines, CG, CHG and CHH, were calculated by dividing the number of non-converted (methylated) cytosines by the total number of cytosines of each type within the sequenced region.

Purified BSP amplification products were ligated to the pGEM-T Easy Vector and transformed into competent JM109 cells using the pGEM-T Easy Vector system I (Promega). White colonies were selected from the ampicillin/X-Gal/IPTG plates, and colony PCR was performed using vector-directed primers to confirm the presence of inserts based on their expected fragment sizes. For plasmid isolation, ten positive colonies from each plate were inoculated into LB medium containing ampicillin. Plasmids containing the target DNA were extracted and purified using the Plasmid Midi Kit (QIAGEN) and then sequenced on an ABI Prism BioDye Terminator version 3.1 sequencing system. Cytosine methylation results were analyzed using the CyMATE online methylation sequence analysis software. Total methylation ratios were calculated by dividing the number of non-converted (methylated) cytosines by the total number of cytosines within the sequenced region; values were expressed as percentages (%). Average methylation levels were expressed as percentage (%) per site for each of the three types of cytosines (CG, CHG, and CHH), and were calculated by dividing the number of non-converted cytosines by the total number of cytosines of each type.