ExoAp-and-Sanger

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Objective

This protocol describes a method for purifying PCR products and preparing them for Sanger sequencing.

Step by step instructions

  1. Prepare a PCR tube for each sample containing approximately 25-50 ng DNA in 10 µl total volume (NFW).
  2. Prepare a master mix containing the following components per reaction. To use, multiply each volume by the number of samples (plus a few to account for pipetting error)
Component per rxn
AP buffer 0.65
Antarctic phosphatase 0.1
Exonuclease I 0.03
NFW 0.22

  1. Add 1 µl master mix to each sample
  2. Incubate 15' at 37°, then 15' at 80°C.
  3. Cool samples on ice, and transfer to 1.5 ml tubes.
  4. Add 1.2 µl of sequencing primer at 10 µM (note this can be either of the primers used for PCR, but NOT both)
  5. Take samples to CGRB (ALS 3012), put them in the "To Drop" box in the freezer, and ask Eli or another lab member to place the order online for sequencing.

History

Created 10:38 Feb 03, 2017 By: Admin

Last updated 09:10 Sep 23, 2019 By: Admin