1. Add the following components into 1.5ml microcentrifuge tube:

   DNA template (100ng)	10µl
   hexanucleotide in 5X reaction buffer	10µl
   deionized water	to 40µl

   Vortex the tube and spin down in a microcentrifuge for 3-5sec. 
   Incubate the tube in a boiling water bath for 5-10min and cool it on ice. Spin down quickly.
    
2. Based on your choice of labeled triphosphate (dATP or dCTP) use Mix A or Mix C , respectively.
    
3. Add the following components in the same tube:

   Mix A (or Mix C)	3µl
   [alpha-32 P]-dATP (or [alpha-32 P]-dCTP)	6µl
   Klenow Fragment, exo- (5u)	1µl

   Shake the tube and spin down in a microcentrifuge for 3-5sec.
   Incubate for 10min at 37°C.
    
4. Add 4µl of dNTP Mix and incubate for 5min at 37°C.
    
5. Stop the reaction by the addition of 1µl 0.5M EDTA, pH 8.0.
    
6. The labeled DNA is used directly for hybridization or stored at -20°C. Removal of the unincorporated label is not necessary for most applications, since the levels of its utilization are usually high. If required, the unincorporated dNTP can be removed by chromatography on Sephadex ® G-50 or by selective precipitation of DNA with ethanol in the presence of ammonium acetate [ 7 ].
    

The percentage of incorporation is determined by DE-81 filter-binding assay.

1. Dilute 1µl of the reaction mix 1:100 with 0.2M EDTA or water. Spot 5µl of it on each of the two Whatman ® DE-81 filters (1.5 x 1.5cm).
    
2. Dry the filters under a heat lamp. Keep one filter aside and use it directly for the determination of total dpm in the sample.
    
3. Wash the other filter 3 times for 5min in 10ml 7.5% (w/v) Na₂ HPO₄ ・12H₂ O for the removal of the unincorporated dNTPs, then once in water and in acetone.
    
4. Dry the washed filter under a heat lamp.
    
5. Transfer and count the filters in an appropriate radioactivity counter.
    
6. The percentage of label incorporation into DNA is defined as below:
    

   incorporated radioactivity (washed filter)
   ------------------------------------------------------------	x 100%
   total radioactivity (unwashed filter)

    

7. Calculate the specific activity of the probe:
   for the calculation of the specific activity of the labeled DNA probe, it is first necessary to evaluate the theoretical amount of DNA generated in the reaction assuming that 100% incorporation of radioactivity occurred:

µCi dNTP added x 4 x 330ng/nmole
-------------------------------------------------------------------	= ng theoret. yield
specific activity dNTP (Ci/mmole= µCi/nmole)

 

330ng/nmole = average molecular weight of a nucleotide.
Further, calculate the percentage incorporation according to DE-81 filter-binding assay results:

dpm incorporated
-----------------------------	x 100% =% incorporation
total dpm

Determine the amount of DNA synthesized:

% incorporation x 0.01 x theoret. yield = ng DNA synthesized

Calculate the specific activity of the product:

total dpm incorp.(dpm incorp. x 20 x 50)
----------------------------------------------------------------	= dpm/µg
(ng DNA synth. + ng input DNA) x 0.001µg/ng

 

 Note  

The factors 20 and 50 are derived while using 5µl of 1:100 dilution for DE-81 filter-binding assay and converting this back to 50µl total reaction volume.

Example. If you use 50µCi (1.85 MBq) [alpha-³² P]-dCTP (3000Ci/mmole) in a standard reaction, the calculation is as follows:

50µCi x 4 x 330 ng/nmole
----------------------------------------	= 22ng theoretical yield
3000µCi/nmole

 

Assuming that 14.91 x 10⁴ dpm remained on the washed DE-81 filter and the unwashed filter retained 18.12 x10⁴ dpm, we recur:

14.91 x 10⁴  
---------------------- x 100% = 82.3% incorporation 
18.12 x 10⁴

0.823 x 22ng = 18.1ng DNA synthesized

Thus, the specific activity is:

14.91 x 104 dpm x 20 x 50
------------------------------------------------	= 1.26 x 109 dpm/µg
(18.1ng + 100ng) x 0.001µg/ng

 

1. Add the following components into 1.5ml microcentrifuge tube:

   DNA template (100ng-1µg)	10µl
   random primer in 5X reaction buffer	10µl
   deionized water	to 42µl

   Vortex the tube and spin down in a microcentrifuge for 3-5sec. 
   Incubate the tube in a boiling water bath for 5-10min and cool it on ice. Spin down quickly.
    
2. Add the following components in the same tube:

   Non-radioactive Labeling Mix (-DIG-dUTP)	5µl
   1mM DIG-dUTP (not included in the kit)	1.75µl
   Klenow Fragment, exo- (5u)	1µl

   Shake the tube and spin down in a microcentrifuge for 3-5sec.
   Incubate for 1 hour at 37°C. Prolonged incubation at 37°C up to 20 hours increases the yield of labeled DNA.
    
3. Stop the reaction by the addition of 1µl 0.5M EDTA, pH 8.0.
    
4. The labeled DNA is used directly for hybridization or stored at -20°C. Removal of the unincorporated label is not necessary for most applications. If required, the unincorporated dNTP can be removed by chromatography on Sephadex ® G-50 or by selective precipitation of DNA with ethanol in the presence of ammonium acetate [ 7 ].

 Note  

1. Using 100ng template DNA can be synthesized about 50-75ng of DIG-labeled DNA after 1 hour incubation and about 240-270ng after 20 hours incubation at 37°C.
2. Other non-radioactive labels (biotin-, fluorescein- dUTP) can be used.

QUALITY CONTROL

All components of the Kit are tested in a labeling reaction of lambda DNA/HindIII fragments, obtained specific activity 1.4x10⁹ dpm/µg DNA.