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)

• ^#For standard and difficult PCR templates, see "PCR Cleanup" below.

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:  Individual Samples vs. 96 Well Plate

A.  Individual Samples

B.  96 Well Plate

PCR Cleanup

Please read this disclaimer about PCR product sequencing:

Sequencing a PCR product is much trickier than sequencing a recombinant plasmid.  High-quality sequence requires high-quality template, but PCR reactions are frequently contaminated with confounding variables (like primers and/or multiple PCR products).  Furthermore, determining the actual concentration of template being targeted for sequencing is complicated by this contamination.

 A.  You clean the PCR product

We highly recommend that you clean the PCR product, because you can thoroughly characterize your sample prior to Sanger sequencing.

• • Confirm there are no primer dimers or confounding PCR products
  • Submit the purified template at the appropriate concentration as suggested HERE
  • Requires very limited handling by DSF
  • Qualifies for discount if 48+ samples

B.  Core cleans the PCR product (i.e. PCR Cleanup and Sequencing) 

We aim to be a high-throughput, low-cost service.  Characterizing PCR Cleanup samples is time- and cost-prohibitive for the DSF.  Nevertheless, our PCR Cleanup SOP does result in quality sequence for many submitted samples.

• • We do not check for absence of primer dimers or confounding PCR products
  • We do not determine template concentration (i.e. we have a 50 uL elution standard)
  • Keeping track of user-submitted primers reduces efficiency of service
  • Inherent variability often requires reruns at cost to customer