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        Plasmid Sequencing

        互联网

        1449

        This protocol gives M13 quality sequence when used with the Quick DNA Plasmid Prep. protocol D.2.

        1、Solutions

        Denaturation Solution
          2 M NaOH 200 ml 10N NaOH
          2 mM EDTA 4 ml 0.5 M EDTA pH 8.0
          up to 1 ml with Q
          store at room temperature

        2、Precipitation Solution

        4M NH4 OAc 530 ml 7.5 M NH4 OAc
          100 mM EDTA 200 ml 0.5 M EDTA pH 8.0
          up to 1 ml with Q
          store at room temperature
          All other reagents are included in the Sequenase™ Version 2.0 kit (USB).

        3、Procedure

        • Combine 13.5 ml of DNA (see protocol D.2) with 1.5 ml Denaturation solution and incubate at 37℃ for 30 minutes.
          • Add 1 ml Precipitation Solution and 75 ml EtOH. Spin for 10 minutes at room temperature.
          • Wash with 80% EtOH and dry for 5 minutes.
          • Resuspend the pellet in 7 ml Q and add 2 ml 5X Reaction buffer and 1 ml of the appropriate oligo at 10 ng/ml.
          • Briefly heat to 65℃ and slow-cool in 50 ml 65℃  water at room temperature (aprox. 30 minutes).
          • Hold on ice and prepare the termination tubes with 2.5 ml of each termination mix.
          • Add the following to the annealed oligo/template:
          1 ml 0.1 M DTT
          2 ml 10X dil. Label mix
          1 ml 2X dil. a 35 S-dATP
          2 ml 8X dil. Sequenase (in TE)
          • Incubate at room temperature for 5 minutes.
          • Add 3.5 ml of the reaction to each termination tube and incubate at 37℃  for 5 minutes.
          • Stop the reaction with 4 ml stop solution and boil prior to loading.

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