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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:
For 24-47 samples, we CAN'T guarantee same day service for individual samples because it slows down our workflow.
PCR Cleanup + Sanger Sequencing
PLEASE NOTE: We offer a "one-size-fits-all" protocol for PCR cleanup with no quantification. Thus, we highly recommend that you clean your PCR product and thoroughly analyze it via gel electrophoresis and fluorometry prior to Sanger sequencing. Recommended concentrations can be found in Template Prep.
Although rare, sometimes DTB and/or our standard Sanger protocol are not sufficient for sequencing a DNA template. We offer custom sequencing for these very difficult templates, which generally involves alternative sequencing chemistry or other modifications to our SOP. We are happy to consult with you regarding custom sequencing at no charge. However, we do charge a higher fee for custom sequencing to cover the cost of the specialized reagents (currently $5.75/sample for UT). Examples:
Problematic secondary structure (such as hairpin loops, stem loops, or palindromic sequences)
Sequencing directly from gDNA
PCR amplicons <200 bp or vectors larger than 15 Kb