Submitting DNA Templates for Sanger Sequencing

Number of samples:

  • Fewer than 21: may be submitted in microfuge tubes.  Tubes should be clearly marked with initials and sample number (eg. AB1, AB2, AB3, etc.).
  • More than 21 samples: must be submitted in a 96 well plate.  Wells A01 and B01 should be left empty.  The plate should be loaded by columns, i.e. fill C01 through H01, then A02 through H02, A03 through H03, etc. 

Template DNA:

  • The templates should be dissolved in Elution Buffer lacking EDTA.  Alternatively, a modified TE buffer with 0.1 mM EDTA can be used.
  • Plasmid DNA:  should be supplied at a concentration of 20-200 ng/µl.  Typically 20‑100 ng is used for each sequencing reaction.
  • PCR Products:  should be supplied at a concentration of  4-20 ng/µl.  Typically 10‑50ng is used for each sequencing reaction.  PCR products must be purified before cycle sequencing to remove excess primers and enzyme.  This service can be provided by AGTC.
  • Cosmids and Fosmids:  should be supplied at a minimum concentration of 200 ng/µl.  Each sequencing reaction requires 1 µg of fosmid/cosmid template.

Primers

  • AGTC has a number of standard primers on hand (see primer list).  If a template-specific primer is required, it should be provided at 5-10 µM concentration.         
  • Note: AGTC uses 3.2 pmol of primer in each sequencing reaction.

Completed sequencing reactions.

  • If sequencing reactions have been performed the client and are being submitted for RUN ONLY, it is best to submit the samples as dry pellets (if ethanol precipitation has been used to clean up sequencing reactions). 
  • If another method of cleanup has been performed, we can add Hi-Di formamide to a final concentration of 25%.