Service Types

Sanger Sequencing

Decision #1:  Standard vs. Difficult vs. Structural 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, with high-quality Phred calls up to 1000 bases.

B.  Difficult Template^#

The Core will add our proprietary Difficult Template Buffer (DTB) to your sample to facilitate processivity of the AmpliTaq DNA polymerase during cycle sequencing.  Common reasons for processivity problems are:

1. • GC-rich templates, with overall greater than 60-70% GC content
   • GC-rich regions, with greater than 60-70% GC content (minimum 10-15 bps)
   • Homopolymers [such as poly(A) tails]
   • Repetitive sequences (such as His tags or other bi- or tri-nucleotide repeats)
   • Low-quality DNA (such as ancient DNA or DNA obtained via forensics)

C.  Structural Template

The Core will add our proprietary HSP Buffer (HSP) to your sample to limit effects of secondary structure on cycle sequencing and capillary electrophoresis of your DNA during sequencing.  Although not often an issue, common secondary structure problems are:

• • Hairpin loops
  • Stem loops
  • Palindromic sequences
  
  ^#For standard and difficult PCR templates, see 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
  • No need to submit sequencing primer separately

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; in fact, we elute all PCR Cleanups in 50 uL of nuclease-free water
  • Keeping track of user-submitted primers reduces efficiency of service
  • Often requires reruns at cost to customer