DNA Sequencing Service Guide

Is your DNA template:

Great?

Is your template pure and confined to a single sharp band (with no additional mysterious bands)?  If it's a plasmid, do you see a sharp single band that produces the correct fragments upon digestion?  Does your plasmid produce a single, sharp band from a PCR with your sequencing primers?  If so, try our Standard Sequencing Service for In-State Academic or Out-Of-State/Commercial customers ($7.50 per sample in-state academic / $9 per sample commercial or out-of-state).  We offer discounts for full plates, and also a half-plate discount for in-state academic customers.

 

For the standard sequencing service, we ask that you provide your DNA template at 15 ng/uL for PCR products and at 50 ng/uL for plasmid, and provide primers at a concentration of 5 uM (i.e. 5 pm/uL, or a 1:20 dilution of 100 uM stock) in a separate tube.  We do provide a number of common sequencing primers at no charge.  We need at least:  2 uL sample for each 250 bases for PCR product, or 10 uL of plasmid per reaction (if you want a template sequenced from forward and reverse directions, then we need double the volume).

 

Very precious, kind of iffy, or there's no time to standardize template concentrations?

If so, we recommend our Full-Service Sequencing procedure.  Full-service sequencing provides more sensitivity than the standard service, and requires less work on your part. Full-service sequencing is $12.50 per sample for in-state customers.

For Full-service sequencing, you should provide us with your DNA template and its concentration along with its primer(s) and its concentration(s). See above for required volumes (note that we will dilute highly concentrated template to the proper titer before sequencing, so the more concentrated your template, the less you need to provide).  Full service sequencing will not salvage sequence from a PCR contaminated with secondary bands, and it won't give you a sequence if your primer doesn't bind to your template.

 

High-GC or Hairpin-containing?

For High-GC/Hairpin-containing DNA templates, we recommend our High-GC Sequencing Service.  This service utilizes an alternate reaction mixture that ameliorates the inhibitory qualities of high-GC and/or heavily-structured sequences that can't be sequenced by standard methods.  High-GC sequencing is $12.50 per sample for in-state customers.  We need DNA templates at a minimum of 5 ng/uL (more is better) and primers at 5 uM (i.e. 5 pm/uL, or 1:20 dilution of 100 uM stock).  The required volumes are as stated above.

 

Do you have other concerns about your template?

Ask us!  We're nice!  Other template concerns include high-AT templates (use the Full-Service option and make a note that your sample is high-AT), high-melting-point primers, problematic plasmids, extraction and purification problems, and many others.  We've seen it all, and can guide you through most difficulties.

For More Information Check Out:

• Troubleshooting Guide - PDF
• Interpreting Electropherograms - PDF

Useful Sites

Some of our favorite tools for use in primer design are free online applications.  Primer3 is an excellent primer design tool.  OligoCalc and OligoAnalyzer are very good tools for determining primer properties, including self-complementarity and heterodimer formation.

 

Want to analyze your sequence?  NCBI's BLASTN suite has it all.

 

Additional DNA Sequencing Information

Our laboratory uses an Applied Biosystems 3730 DNA analyzer that can process 48 samples per run.  At approximately 2 hours per sequencing run, we can process up to 576 individual samples in a 24-hour period.  We offer a number of sequencing services to reliably sequence your template regardless of it base composition.

We consistently deliver excellent sequence quality and read lengths (over 900 bases), and our sequence QC methods provides an account of the quality of your sequences and suggestions to improve low-performing samples.

Our Fragment Analysis service provides the platform for running a number of SNP, AFLP, microsatellite, and other variant-detection protocols.  We are capable of analyzing 48 individual samples per run, and most runs are complete within an hour.  Most runs utilize standard instrument protocols, but we are able to develop protocols to suit your analytical needs.

We recommend using ABI sizing standards and end-labeled oligos for most fragment analysis, but we are very happy to meet with you to develop your strategy.