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UVP Minidizer Hybridization Oven 

 

12 

81-0169-02 Rev J

 

 
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 

 

 
 
 

 

 

Summary of Contents for 95-0330-01

Page 1: ...________________________________________ Analytik Jena US 2066 W 11th Street Upland CA 91786 Tel 909 946 3197 Fax 909 946 3597 Web Site www us analytik jena com Ultra Violet Products Ltd Unit 1 Trinit...

Page 2: ...UVP Minidizer Hybridization Oven 2 81 0169 02 Rev J Table of Contents Introduction 3 Hybridization Oven Specifications 4 Installation 5 Operation 6 Service Procedures 6 Hybridization Techniques 10...

Page 3: ...er at 68 C is 0 1 C at 55 C is 0 1 C and at 42 C is 0 1 C WARNING There may be build up of pressure within the hybridization bottles when they are taken from ambient to hybridization temperature To he...

Page 4: ...ber Volts Hz 95 0330 01 115V 60Hz 95 0330 02 230V 50Hz 95 0330 03 100V 50Hz Specifications Net Weight 11 3 lbs 5 kg Temperature Ambient 10 C to 80 C Heating Element 500 watts Temperature Display LCD R...

Page 5: ...to the carousel gently press them into the clips 2 To insert bottle holder begin by placing the large end of the bottle holder into the slotted holder on the right wall Twist until the shaft locks int...

Page 6: ...Temperature Setpoint The current setpoint value can be altered using the UP and DOWN buttons while the setpoint is being displayed To change the setpoint from normal mode proceed as follows 1 Press t...

Page 7: ...card the bottle The bottles should not be used at temperatures above 70 C Decontamination Bottles and Caps Soak items in a diluted detergent solution overnight Remove from detergent and rinse items wi...

Page 8: ...s Authorization RGA number must be obtained from AJ s Customer Service prior to returning any product If you are in North America South America East Asia or Australia If you are in Europe Africa the M...

Page 9: ...all apply to any instrument or part thereof that has been subject to accident negligence alteration abuse or misuse by the end user Moreover AJ makes no warranties whatsoever with respect to parts not...

Page 10: ...nisms of Nucleic Hybridization Hybridization occurs with a process called nucleation whereby the two separate nucleic acid strands come into close proximity of each other A duplex region is formed whe...

Page 11: ...s solution Unacceptable high background Use less probe Hybridize at lower salt higher temperature Wash with lower salt higher temperature Incubate with very low salt change nuclease solution Use a sma...

Page 12: ...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...

Page 13: ...ng Water Bath Bucket of ice Gloves Plexiglas shield UVP Minidizer HybriCycler or Hybridizer Hybridization Oven Procedure 1 Add 15 mL of prehybridization solution to each hybridization bottle containin...

Page 14: ...nal wash dry blot on filter paper for 10 minutes This is a good time to quickly pass your hand held radioisotope reader beta or gamma counter over your blot to get a general idea as to the exposure ti...

Page 15: ...Membrane following hybridization Procedure 1 Mix equal volumes of detection reagents 1 and 2 2 Pipette the mixture over the surface of the membrane and leave at room temperature for 1 minute 3 Drain t...

Page 16: ...UVP Minidizer Hybridization Oven 16 81 0169 02 Rev J...

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