Protocol S.1

Plasmid Sequencing

 

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

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

Precipitation Solution

4M NH4OAc 530 ml 7.5 M NH4OAc

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).

 

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. a35S-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.