UVP HB-500 Minidizer - Manual (Page 12)

HB-500 Minidizer Hybridization Oven 12
Protocol 1:
Random Priming Method for Tagging DNA with Fluorescein-Labeled Nucleotide
and Others
This method uses DNA polymerase to incorporate Fluorescene-11- dUTP into double stranded DNA probes.
This protocol can be used to incorporate any tagged nucleotides.
Equipment
•
Micropipettes and tips
•
Boiling water bath
•
1.5 mL Microcentrifuge tubes
•
Microcentrifuge
•
Cap lock for Microcentrifuge tube
•
Water bath set to 37°C
Reagents
•
Deionized, sterile water
•
EDTA, 0.5 M
•
Klenow DNA polymerase , 4-
5 units/μL
• Nucleotide mix (300μm each of dAT P, dCTP, dGTP and 60μm dTTP)
•
Random nonamer (9-mer) primers,
2.5 μg/μL in water
•
Reaction buffer, 10X: 50mM MgCl2, 10mM 2-Mercaptoethanol, 500 mM Tris-HCl, pH 7.5\
•
Tagged nucleotide: fluorescene-11-dUTP
•
Template DNA in water (5ng/ mL)
Procedure
1. Pipette 10 mL of template DNA plus 10 mL of water into a microcentrifuge tube and cap tightly.
Cover cap with a cap lock or bend a paper clip in half and secure over the microcentrifuge tube.
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 mL Nucleotide mix
b. 5 mL Tagged nucleotide
c. 5 mL Reaction buffer (x10)
d. 5 mL Random primers
e. 10 mL Boiled DNA
f. 14 mL Water
g. 1 mL DNA polymerase
h. Mix gently and incubate at 37 °C for 1 hour
i. Stop the reaction by adding 2 mL EDTA
j. Store probes at -20 °C in the dark