Thursday, November 20, 2003

Description
Bst DNA polymerase-catalyzed radiolabeled two-step sequencing reactions For single-stranded DNA sequencing

Procedure
1. Prepare the following extension reaction in a microcentrifuge tube:

750 ng M13 template DNA
2 ul Bst reaction buffer
2 ul Bst nucleotide extension mix
1 ul oligonucleotide primer (2.5 ng/ul)
0.5-1 ul [alpha]-32-P-dATP or [alpha]-35-S-dATP
1 ul diluted Bst polymerase (0.1 U/ul)
q.s. sterile ddH2O
12 ul

[alpha]-32-P-dATP (PB 10384) and [alpha]-35-S-dATP (SJ 1304) from Amersham.
Dilute the Bst polymerase (BioRad 170-3406) in Bst dilution buffer.

2. Incubate the reactions for 2 minutes at 67degC, and briefly centrifuge to reclaim condensation.

3. Remove 2.5 ul aliquots for each reaction into the four base-specific termination mixes (either short or long), already pipetted into a V-bottomed microtiter plate (Dynatech).

4. Incubate the reactions for 10 minutes at 67degC, and briefly centrifuge to reclaim condensation. It is possible to store the reactions at -70degC at this stage.

5. Stop the reactions by the addition of 4 ul of agarose gel loading dye and incubate for 5-7 minutes at 100degC.



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