DNA polymerase pre-steady state pyrophosphorylation kinetics

1. Materials

Enzyme-DNA solution (1 mL)

300 nM ³²P-primer/450 nM template.
100 nM KF^-
50 mM Tris-HCl (pH 7.5)
2.5 mM EDTA
200 g/mL BSA

dNTP-MgCl₂ (1 mL)

0 to 500 mM dNTP
50 mM Tris-HCl (pH 7.5)
12.5 mM MgCl₂

HCl Quench

1.3 N HCl

EDTA Quench

300 mM EDTA (pH 8.0)

Formamide-dye solution

100 mL formamide
25 mg bromophenol blue
25 mg xylene cyanol

2. Pre-Steady-state kinetics

1. Pre-equilibrate system to 25 °C.
2. Load reaction buffer without MgCl₂, EDTA, or BSA (eg 50 mM Tris (pH 7.5)) into side pistons.
3. Load Quench into middle piston
4. Load enzyme-DNA to right sample loop
5. Load dNTP-MgCl₂ to left sample loop
6. Typical reaction times
   

   Time point	sec
   1	0.002
   2	0.004
   3	0.007
   4	0.01
   5	0.015
   6	0.02
   7	0.03
   8	0.05
   9	0.075
   10	0.1
   11	0.15
   12	0.2
   13	0.3
   14	0.5
   15	0.75
   16	1.0
   17	2.0
   18	5.0
   19	10.0
   20	20.0
   21	30.0
   22	50.0

7. Quench
   1. with EDTA.
   2. with HCl. After 30 sec neutralize by adding 100 L 2M Tris, 0.1 M EDTA
8. Add 100m L formamide-dye solution.
9. Load onto PAGE (15% acrylamide)
10. Run for 2 h at 200V
11. Quantitate with phosphorimager