UVP HybriCycler - Manual (Page 11)

HC-3000 HybriCycler Hybridization Oven 11
Protocol 1: Random priming method for tagging DNA with fluorescein-lebeled
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 (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: 50mM MgCl
2
, 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. Immeadiately 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:
6. 10 mL Nucleotide mix
7. 5 mL Tagged nucleotide
8. 5 mL Reaction buffer (x10)
9. 5 mL Random primers
10. 10 mL Boiled DNA
11. 14 mL Water