Sanger Sequencing

(under construction)

  • ExoSAP
  • Sequencing reaction mix
  • UH, Manoa Sequencing Service

The ExoSAP reaction "removes" the dNTP's and PCR primers from the PCR product to prepare it for seqeuncing.  It contains an exonuclease and shrimp alkaline phosphatase.

  • 10 ul PCR product
  • 4 ul ExoSAP

The mixture is heated to 37C for 15 minutes then 80C for 15 minutes.  It is under the program "EXSAP" on the thermocycler.

The sequencing reaction mix for a 10ul volume is

  • 3ul H2O
  • 3ul ExoSAP'd PCR product
  • 4ul 1uM sequencing primer

We use the University's sequencing service (link), which is currently $2.50 per lane.  This form (link) must be filled out and the plate and form dropped off at Snyder 312.  We put the reaction for each sample in a small eppendorf tube arranged in order in a plate.

Ordering: (not an endorsement, links to company products are put here for convenience; this list is incomplete and we have not tried and compared many competitive products)

ExoSAP-IT from Affymetrix

(to be continued...)

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