Dear Sir. Concerning your issue about the protocol of the manufacturer of X-Terminator and Big Dye Sequencing . The BigDye XTerminator® Purification Kit is a fast, simple purification method for DNA sequencing reactions that removes unincorporated BigDye® terminators and salts. No more dye blobs! Cleanup is complete in under 40 minutes and requires less than 10 minutes of labor. • Eliminate liquid transfer – Use a single plate. • Obtain complete dye blob removal. • Stabilize the samples before analysis. • Improve your sequencing workflow. • Perform manual or automated protocols. • Get rapid, reliable, and reproducible results. Simple Purification Process Traditional purification methods, such as ethanol precipitation, require the addition of multiple reagents along with decanting and centrifuging steps. The BigDye XTerminator® Purification Kit requires the addition of only two reagents which can be added sequentially or premixed: • XTerminator™ Solution – Scavenges unincorporated dye terminators and free salts from the post-sequencing reaction. • SAM™ Solution – Enhances the performance of the XTerminator™ Solution and stabilizes the post-purification reactions. The protocol is very simple (see figure 1): 1. Perform cycle sequencing with BigDye® terminators. 2. After cycle sequencing, centrifuge the reaction plate briefly, and then pipette the correct volume of SAM™ Solution into each plate well. 3. Pipette the correct volume of XTerminator™ Solution into each well: 4. Vortex the XTerminator™ Solution bulk container briefly. 5. Using a wide-bore pipette tip (recommended wide-bore tips), pipette the correct volume of liquid into each plate well 6. Seal the plate using a heat seal or Clear Adhesive Film (P⁄N 4306311). Vortex for 30 minutes (recommended 96-well microplate vortexers and 384-well microplage vortexers), then centrifuge the reaction plate briefly. 7. Place the reaction plate in the Applied Biosystems DNA analyzer. Select run module and run plate. Excellent Long Fragment Recovery You won’t sacrifice sequence length when you use the BigDye XTerminator® Purification Kit. Figure 2 shows data from the DNA Technology Unit, Plant Biotechnology Institute (NRC-PBI) Saskatoon, Canada. The Q20 base read=1044 bp. Improved Short Fragment Recovery Use the BigDye XTerminator® Purification Kit to obtain higher quality data. Figure 3 shows a comparison between the BigDye XTerminator® Purification Kit and ethanol precipitation-based purification performed at the DNA Technology Unit, Plant Biotechnology Institute (NRC-PBI) Saskatoon, Canada. The BigDye XTerminator® Purification Kit results in superior short fragment recovery. Ethanol purification produces sequence data marred by dye blobs. Increased Stability Sealed reaction plates are stable for 48 hours at room temperature or 10 days at 4°C You can re-run the same sample in case of a power interruption, computer malfunction, etc. without having to repeat the experiment. This increases your flexibility when loading a reaction plate on a CE instrument. Run Modules Available Applied Biosystems provides download-able run modules for use with the BigDye XTerminator® Purification Kit and Data Collection Software. These modules are designed for the 3100, 3100–Avant (with Data Collection v2.0 or above), 3130⁄3130xl, and 3730⁄3730xl Analyzers. The software modules for the BigDye XTerminator® Purification Kit are compatible with the Windows® 2000 and Windows® XP operating systems and are available on our software download page: http:⁄⁄www.appliedbiosystems.com⁄absite⁄us⁄en⁄home⁄support⁄software⁄dna-sequencing⁄bigdye-xterm-purif-kit.htm. I think the following below links may help you in your analysis:

https://www.thermofisher.com/order/catalog/product/4376486

http://www3.appliedbiosystems.com/cms/groups/mcb_support/documents/generaldocuments/cms_042772.pdf

https://www.griffith.edu.au/__data/assets/pdf_file/0025/268450/BigDye-XTerminator-Protocol.pdf

Thanks