Sanger Sequencing
Decision #1: Standard vs. Difficult Template
A. Standard Template
Most plasmid samples would be considered standard. Our Sanger sequencing SOP (standard operating procedure) is designed to work with miniprep-quality recombinant plasmid DNA and a high-specificity sequencing primer to give contiguous reads >800 bases and over 1000 high-quality (Q>20) Phred calls
B. Difficult Template
We will add our proprietary Difficult Template Buffer (DTB) to your sample to facilitate template denaturation and processivity of the AmpliTaq DNA polymerase during cycle sequencing. Common reasons for processivity problems are:
- GC-rich templates, with overall greater than 60-65% GC content
- GC-rich regions, with greater than 60-65% GC content (minimum 100-150 bp)
- Homopolymers [such as poly(A) tails]
- Repetitive sequences (di- or tri-nucleotide repeats, such as His tags)
Decision #2: My Primer vs. Core Primer
A. My Primer
You supply a primer for cycle sequencing. YOU MUST ADD THE PRIMER TO YOUR SAMPLE (aka pre-mixed).
We reserve the right to delay or refuse orders with the primer supplied separately.
B. Core Primer
We can supply any of these 10 common MCS primers for cycle sequencing: