Hoefer HB1000 - Instrukcja (Strona 25)

•
p16
Reagents
• Deionized, sterile water
• EDTA, 0.5 M
• DNA polymerase, Klenow fragment, 4 – 5 units/μL
• Nucleotide mix (300 μM each of dATP, dCTP,
dGTP and 60 μM dTTP)
• Random nonamer (9-mer) primers,
2.5 μg/μL in water
• Reaction buffer, 10X (50 mM MgCl
2
,
10 mM 2-mercaptoethanol,
500 mM Tris-HCl, pH 7.5)
• Tagged nucleotide: fluorescein-11-dUTP
• Template DNA in water (5 ng/mL)
Procedure
1. Pipette 10 μL of template DNA plus 10 μL of
water into a microcentrifuge tube and cap tightly.
Secure cap with a cap lock or use a bent paper-
clip to ensure that the cap does not pop when the
contents are boiled.
2. Place the tube into the boiling water bath for
5 minutes.
3. Immediately place tube on ice for 5 minutes.
4. Centrifuge for 15 seconds in microcentrifuge.
5. Add the reagents listed below to a fresh tube
on ice in the following order:
a. 10 μL Nucleotide mix
b. 5 μL Tagged nucleotide
c. 5 μL Reaction buffer (10X)
d. 5 μL Random primers
e. 10 μL Boiled DNA
f. 14 μL Water
g. 1 μL DNA polymerase, Klenow fragment
6. Mix gently and incubate at 37 °C for 1 hour.
7. Stop the reaction by adding 2 μL EDTA.
8. Store probes at -20 °C in the dark.