Service Types

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

The Core 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:

1. • 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

B.  Core Primer

We can supply any of these 10 common MCS primers for cycle sequencing:

Decision #3:  Tubes vs. 96-Well Plate

A.  Tubes

B.  96-Well Plate

Custom Sequencing

A.  Very Difficult Templates

We offer custom sequencing for very difficult templates, which generally involves alternative sequencing chemistry or other modifications to our standard Sanger protocol.  For example, we can attempt to sequence gDNA or templates with problematic secondary structure (such as hairpin loops, stem loops, or palindromic sequences).  We recommend trying "difficult template" service first (see above), but you can certainly request a specific chemistry directly by choosing "custom sequencing" as the service type, and indicating any additives and/or modifications.  We are happy to consult with you regarding custom sequencing, which is currently at no charge.

B.  Custom Projects

We also offer custom projects, including experimental design.  Such projects require consultation and an outline of project goals and deliverables.  The projects are billed at an hourly rate + consumables (primarily reagents).  Our current hourly rate is $32/hour.  For example, we can do CRISPR or SNP analysis, including any cloning, DNA purification, PCR, qPCR, and sequencing. 

PCR Cleanup

PLEASE NOTE: Sequencing a PCR product is much trickier than sequencing a recombinant plasmid because:  

1. PCR reactions are frequently contaminated with confounding variables like primers and/or multiple PCR products
2. PCR product yields are more variable than minipreps 

Essentially, we offer a one-size-fits-all protocol for PCR cleanup with no quantification.  The inherent variability among PCR samples often requires optimization (via reruns) at cost to the customer.  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.  If you really prefer to have us clean the PCR, we recommend the following relationship between amplicon size and cycle number: 

•  200-500 bp amplicons, 12-15 cycles of PCR
• 500-1000 bp amplicons, 15-18 cycles of PCR
• >1000 bp amplicons, 18-22 cycles of PCR