There are three things to keep in mind when kinasing DNA. First, the 5' ends of the DNA must be dephosphorylated. This is accomplished by using alkaline phosphatase. We used to use Calf Alkaline Phosphatase but now we use Shrimp Alkaline Phosphatase. The latter is more readily inactivated by heat. Second, the ATP concentration in the kinase reaction must stay at or above 1 uM through-out the reaction. This is the Km of the kinase. To maintain an appropriate level of ATP, the reactions are performed in 5 to 10 ul and the amount of DNA is kept low enough so ATP stays in excess. Finally, you should have well-characterized DNA. Know the concentration or pmoles of ends so that the reaction doesn't reduce the ATP below 1 uM. Check a portion of the DNA on a minigel before kinasing so that you know that it is intact and at the suspected concentration.

The following description is for preparing an end-labeled fragment of about 300 bp that is part of a pUC13 subclone. This could be an hsp70 promoter fragment that will be used for footprinting. Such a plasmid is about 3 kb long, and 1 ug corresponds to about 1 pmole of ends (two ends per linear DNA fragment). The recommendations can be easily adjusted for different sizes of DNA by calculating the pmoles of ends.

Step 1: First cut and alkaline phosphatase treatment.

1. Cut the the DNA with the first enzyme. You will need 1 to 3 ug for a kinase reaction. I typically prepare 10 ug or more of DNA so I have enough for several labellings. Check 50 ng of the DNA on a minigel at the end of the digest to be certain that it has cut to completion.
2. Ethanol precipitate the DNA by adding 1/10 volume of 3M NaOAc pH 6.0 and 2.5 volumes of Ethanol. Mix thoroughly, place on ice for at least 15 minutes, and microfuge in the cold for at least 20 minutes. Remove the supernatant and add 100 ul of cold 75% ethanol. Microfuge for 5' in the cold. Remove supernatant and allow the sample to air dry.
3. For SAP treatment, I treat 20 ug with 1unit for 1hr at 37°C. The reaction is done in 40 ul using the manufacturer's buffer. Then inactivate the SAP by heating the sample to 65°C for 25'.
4. Ethanol precipitate as described above. Dissolve DNA in TE at an anticipated concentration of 1 ug/ul. You may want to obtain a more accurate measure of the DNA concentration using the fluorometer.

Step 2: Kinasing.

1. Take out the stock of gamma 32P-ATP (stock at >7000 Ci/mmole, ~150 uCi/ul) and put it behind a shield in the radioactive area. Check the label carefully to make sure you have the right stuff. Allow 20' to 30' for the nucleotide to thaw. This is important as a frozen ice chip of radioactivity can be easily ejected from the container when you go to remove some ATP from the stock.
2. Combine in a 1.5 ml tube:
1-3 ug of restriction-cut, dephosphorylated DNA (see step 1).
0.5 ul of 10 X kinase buffer.
Enough dH₂O to give a volume of 3.8 ul.
Mix gently
3. All subsequent manipulations should be done in the radioactive area behind a shield. Wear two layers of gloves and work with extreme caution. Constantly check for contamination using the Geiger counter set on the most sensitive scale. If you contaminate the outside of the tube in which the reaction is being performed, transfer the contents to a new tube. Most surface contaminations can be removed by wiping the contaminated surfaces with damp paper towels. Contaminated materials should be discarded in the radioactive trash. Radioactive material should never be discarded in the regular trash, otherwise use of radioactivity may be curtailed.
4. Very carefully add 1 ul of gamma 32P-ATP (stock at >7000 Ci/mmole, ~150 uCi/ul) to the mixture in the 1.5 ml tube. When removing radioactivity from the stock or adding the radioactivity to the sample, avoid contacting the pipette tip with the sides or lids of the container. After expelling the radioactivity from the tip, carefully remove the tip with your free hand. Do not use the ejector since the negative pressure generated during ejection can cause contamination of the pipette tip.
5. Mix the sample by gently tapping the side of the tube. Avoid splashing the radioactivity on the upper surface of the tube.
6. Add 0.2 ul of kinase using intramedic tubing attached to a 1 ul syringe. Use a piece of tubing that is about 4 cm long so you can easily reach the bottom of the tube. Mix the kinase into the sample by stirring the tip of the tubing while the kinase is expelled.
7. After expelling the kinase, discard the radioactive tubing in the radioactive waste.
8. Mix the sample by gently tapping the tube.
9. Incubate for 30' at 37^oC. This incubation should be done in the heating block in the radioactive area if possible.
10. Add 20 ul of TE and heat inactivate the kinase by incubating the sample at 65°C for 15'.
11. Add 2.5 ul of 3M NaOAc pH 6 (1/10 volume) and 62.5 ul of 100% EtOH (stock stored at -20^oC, 2.5 volumes). Carefully mix the sample but avoid splashing the radioactivity up onto the upper region of the tube. Look carefully for the elimination of schlieren lines as an indication that the sample is mixed.
12. Incubate the sample on ice for 15' and then centrifuge for 20' at 4^oC in the microfuge.
13. Carefully remove the supernatant with a pipette and temporarily discard the radioactive supernatant in the radioactive waste bottle in the radioactive area.
14. Add 100 ul of 75% Ethanol (-20^oC) to the DNA pellet, gently agitate the tube, centrifuge for 5' at 4^oC. Remove as much of the wash as possible and temporarily discard it in radioactive waste bottle.
15. Air-dry the DNA by setting the tube on its side behind the radioactive shield and waiting 15 to 30'.
16. While the DNA is drying, transfer the liquid radioactive waste from the small bottle to the large radioactive waste bottle usually located under the sink behind the radioactive area. Assume that all of the radioactivity used in the labelling ends up in the liquid waste and record this amount on the tag.
17. The dried DNA pellet is now ready to be restriction cut in a volume of 20 ul and the desired restriction fragment can be purified on a thin acrylamide gel containing TBE as running buffer.

Step 3: Second restriction enzyme digest.

1. Digest the radioactively labelled DNA in a total volume of 20 ul. At the end of the digestion period, stop the reaction by adding 6 ul of 6X ficoll loading dye (30% ficoll, 100 mM EDTA pH 7.6, 0.02% Bromophenol blue). It is important to use ficoll rather than glycerol or sucrose since this somehow keeps the DNA from streaking when it is run on the preparative acrylamide gel.
2. The sample can be stored at -20 overnight or run immediately on a thin acrylamide gel.

Step 4: Gel purification of radioactive fragment.

The DNA should be in Ficoll buffer. 6X = 30% ficoll, 100 mM EDTA pH 7.6, 0.02% Bromophenol blue. This loading buffer produces bands that a markedly sharper than sucrose or glycerol buffers. The 6X ficoll buffer can be added directly to the sample after enzyme treatments without prior precipitation of the DNA.

Prepare a 0.2 mm thick acrylamide gel according to a recipe below. The recipes are for 20 cm x 20 cm gels. Siliconize one plate so that the gel sticks only to one side when the plates are separated. Wipe the other side with ethanol; this helps prevent air bubbles from getting trapped between the plates as the gel is being poured. Typically, the wells are about 2 to 3 cm wide.
 

	6%	8%	10%
29.2% Acrylamide/0.8% Bis	3 ml	4 ml	5 ml
10X TBE	1.5 ml	1.5 ml	1.5 ml
Water	10.5 ml	9.5 ml	8.5 ml
10% Ammonium persulfate	105 ul	105 ul	105 ul
Gently swirl solution before adding TEMED
TEMED	11.25 ul	11.25 ul	11.25 ul

Assemble the gel in the apparatus, rinse the wells and then carefully load the samples.  There is no need to pre-run the gel.  Run the gel at 150 to 200 volts.

Isolation of a radioactive fragment: Be sure to keep track of the radioactivity with a geiger counter. The electrophoresis buffer and gel will be very radioactive and the radioactivity must be appropriatedly dealt with. Separate the glass plates so the gel remains attached to one plate. Wrap the gel and plate in plastic. In the dark, lay a piece of X-ray film over the gel. Mark the edges with a marker so the film can be oriented on the gel. Overlay the film with a sheet of plexiglass so the film stays flat and expose for 3 to 5 minutes. Develope the film and cut out the portion of film corresponding to the desired radioactive band. Lay the film on the gel in the proper orientation and use the film as a template to cut out the region of the gel with a scapel. Remove the film and then use tweezers to remove the small piece of plastic wrap covering the gel slice. Note that the plastic wrap must not be transferred along with the gel slice because the DNA will adher to it. Transfer the gel slice to a siliconized tube. Add 400 ul of 0.1 M NaCl, 10 mM Tris pH 8, 1 mM EDTA and incubate the tube overnight at 37°C. You can improve the yield by adding 1 ug of carrier DNA such as HaeIII-cut E. coli DNA but good yields can be obtained without carrier DNA probably because linear acrylamide leached from the gel is excellent DNA carrier. If you choose to use a carrier, make certain that it won't interfer with subsequent uses of the radioactive DNA.

The next day, centrifuge the sample for a few minutes. Carefully transfer the solution to a fresh siliconized tube; avoid transferring any pieces of gel. The majority of the radioactivity should transfer, but some residual counts will remain with the gel slice. Add 40 ul of 3 M NaOAc pH 6 and 1 ml of Ethanol. Ice for 30' and centrifuge in the cold for 30'. Remove the supernatant; most of the radioactivity should remain behind in the tube. Add 100 ul of 75% cold Ethanol and centrifuge for 5' in the cold. Remove the supernatant. Air-dry the DNA and then dissolve in 10 to 50 ul. Determine the amount of radioactivity in 1 ul.