Method: Using Boehringer Mannheim (BMB) Random Primed DNA Labeling Kit

June 27, 1990

Srini Ramachandra

Principle:

This is an alternate method to the other method of Primer Extension labeling. This protocol exclusively uses reagents supplied with the BMB kit. Double stranded DNA is heat-denatured andrandom hexanucleotide primers are attached to the single strand. The synthesis is carried out using Klenow fragment of DNA polymeraseI incorporating 32P-dCTP at the same time. The Klenowenzyme lacks the 5'----> 3' exonuclease activity, so that the radioactive product is synthesised exclusively by primer extension rather than nick translation. The reaction is carried out at pH6.6 thereby reducing the 3' ----> 5' exonuclease activity of the Klenow enzyme. On an average 35-40% incorporation of 32P-dCTP can be expected. A 25 ng reaction will usually generate 5 x 107 dpm.Ideally we need 1 - 5 x 107dpm of labeled probe DNA per blot and 0.5 - 2.5 x 107dpm of labeled lambda DNA per blot. If more than 5 blots are used per probe you can double or triple all components of the reaction, but you will have to reduce the volume of TE applied to the column in order to maintain the volume of labeled probe at or below 500 µl.

Time Required:

2 hours

Special reagents required:

• BMB Random Primed DNA Labeling Kit (BMB Cat.# 1004 760)

Procedure:

Labeling the probe:

Note: The volumes given below are for a 25 ng reaction. These volumes can be scaled up to 3 times for a 75 ng reaction.

1. Thaw out 32P-dCTP on a fresh piece of absorbent diaper paper behind the radioactive shieldfor at least 30 minutes. (But do not let it sit at room temperature for hours, as the nucleotide will start to breakdown).
2. Prepare 12.5 ng/ul dilutions of probe DNA in TE and store the excess at 4 degrees C or -20 degrees C for future use.
3. For each reaction add 2 µl of probe DNA (25 ng total ) to a sterile microcentrifuge tube.
4. Add 7 µl of sterile dH2O (total volume = 9 µl).
5. Clamp the tubes in a metal holder and denature DNA in a boiling waterbath for 2-10 minutes. If using the Fry-daddy, unplug it occasionally to maintain an approximate temperature of 95 degrees C. [You can also denature the probe DNA in a beaker (glass) containing water and boiling it over a bunsen burner flame for 2-10 minutes].
6. Quick-cool the tubes on ice (~2 minutes). Remove tubes from the metal holder. Touch-spin the tubes in a table top microcentrifuge to collect the condensation from the sides.
7. Add 3 µl of a 1:1:1 mixture of cold dATP, dGTP and dTTP (AGT combo) to each tube.
8. Add 2 µl of reaction mix #6 (BMB kit) to each tube. Mix gently.
9. Add 5 µl of 32P-dCTP and 1µl of Klenow enzyme to each tube, mix well. Work behind a plexiglass shield in the radioactive work area.
10. Incubate at 37 degrees C in a heat block behind a shield for 30 minutes.

Follow the other method: "Labeling DNA with 32P-dCTP by Primer Extension" for the rest of the steps which include P-60 column preparation and probe purification.

Solutions:

Refer to the other method: "Labeling DNA with 32P-dCTP by Primer Extension"

References:

Feinberg, A.P. and B. Vogelstein, B. (1983). "A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity". Anal. Biochem. 132: p. 6-13.

Boehringer Mannheim Biochemicals: Random Primed DNA Labeling Kit Protocol.