Plasmid Sequencing

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.