Hybridization Ovens
Page 21
81-0169-01 Rev Q
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:
1.
10 mL Nucleotide mix
2.
5 mL Tagged nucleotide
3.
5 mL Reaction buffer (x10)
4.
5 mL Random primers
5.
10 mL Boiled DNA
6.
14 mL Water
7.
1 mL DNA polymerase
8.
Mix gently and incubate at 37 °C for 1 hour
9.
Stop the reaction by adding 2 mL EDTA
10. Store probes at -20 °C in the dark
