Method: Primer Extension Labeling of DNA in Low Melt Agarose

4/18/1990

C. Helms

Purpose:

This procedure allows a restriction fragment(s) to be labeled directly from a gel slice, without the associated losses of DNA usually seen with gel purification methods. Often the best hybridization results are obtained using a fragment of the cloned probe DNA (the signal will be stronger when a fragment specific to the target DNA is labeled rather than the entire clone). The procedure can also be used to obtain results from a blot known to contain contaminating vector sequences because only the clone insert would be used as probe.

Special reagents:

• FMC Seaplaque Low Melting Temperature agarose

Time required:

2 hours- overnight

Procedure:

1. Restrict clone DNA and run a low melt agarose gel in TA buffer + ethidium bromide. A minigel can be run if the fragments can be separated adequately. Excise the band using long wavelength UV lightbox and a clean razor blade. Trim the gel slice of as much extra agarose as possible before placing in an eppendorf tube.
2. Melt the gel slice at 70 degrees C for 5- 15 minutes, remove 0.1 µg (up to 34 µl) and place in a fresh tube. Often it is difficult to estimate concentrations. However if you try to stay close to or below 0.1 µg of DNA per reaction, the specific activity of the probe will be high. Add sterile dH2O to bring the volume to 34 µl. Place in the boiling water bath for 5 minutes to denature the probe. Place in a 37 degrees C waterbath or heatblock and quickly add the following:

   2.0 µl	OL solution primer (1mg/ml pd(N)6 ), see primer extension protocol
   5.0 µl	10X salts (recipe below)
   2.5 µl	20 X dNTPs - C (recipe below)
   1.0 µl	BSA (10 mg/ml)
   5.0 µl	32P dCTP
   0.5 µl	Klenow fragment
   ------------------
   16 µl	total volume added

   Mix well by pipetting up and down with a pipetman.

3. Incubate at room temperature for 3 hours to overnight, or at 37 degrees C for 1-2 hours. Stop the reaction with 40 µl Labeling Stop Mix and put at 65 degrees C for about 10 minutes until the mixture is liquified.
4. Warm up the TE used to elute the probe from the P60 column in a 50 degrees C incubator, and prerinse the column with 1 ml of the warmed TE. Apply the probe to the column as usual, but be sure to begin the elution as soon as the probe mix has entered the column to prevent the agarose from solidifying on the column, and work with one column at a time. Collect the 500 µl containing the light blue (dextran blue) dye. Count the probe and use as usual.

Solutions:

• 10X Salts

  10X Salts	Final Concentration
  250 µl 2M Tris-HCl pH 8.0	0.5 M Tris-HCl pH 8.0
  200 µl 100 mM DTT	20 mM DTT
  50 µl 1 M MgCl2	50 mM MgCl2
  400 µl 1 M KCl	0.4 M KCl
  100 µl dH2O

  Store at -20 degrees C.

• 20X dNTP-C (10 mM dNTP-C):

  Find dATP, dGTP & dTTP in the Ultrapure dNTP set (Pharmacia, Cat.#27-2035-01). They are supplied in 100mM concentrations.

  Combine 100 µl of each nucleotide in an eppendorf tube and add 700 µl dH2O. This will make a 10 mM stock of dATP, dGTP, dTTP = 20X dNTP-C.

  Store all stock solutions at -20 degrees C.

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

Feinberg, A. P., and B. Vogelstein. (1984) " Addendum: A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity." Anal. Biochem. 137: 266.

Struhl, K. (1985). "A rapid method for creating recombinant DNA molecules". BioTechniques 3: 452-453.

CRI Laboratory Manual: RFLPs Project (1989).