Labeling of DNA Probes using Primer

Tuesday, October 21, 2003

Description
This technique is an alternative to labeling by nick translation. It is less fussy and generally gives considerably hotter probes. It has the additional advantage of working with even quite dirty DNA preps

Procedure
1. Boil 20 to 40 ?g target DNA in 32 ?l of ddH2O for 5 to 10 minutes and then snap cool the DNA in an ice bath (See Hints #3 and #4).

2. Set up the labeling reaction as follows:

32 ?l template DNA

10 ?l of 5X Oligo Labeling Buffer

2 ?l of 10 mg/ml Bovine Serum Albumin

5 ?l of ?-[32P]-dATP (3,000 Ci/mmol)

1 ?l (2 to 5 Units) Klenow fragment

3. Incubate the reaction at room temperature for 3 to 4 hour. Most of the incorporation has occurred by 1 hour and continues to until about 3 or 4 hours (see Hint #5). Unlike nick translation, it does not then diminish.

4. Separate the unincorporated nucleotides using small spin columns in 0.5 ml microcentrifuge tubes or other preferred method.

5. Incorporation of radiolabel into the oligonucleotides is usually between 50% and 80%, unless you start with less than 20 ng to 30 ng DNA. The specific activity can reach about 3 billion dpm/ug DNA.

Recipes
?-[32P]-dATP

?-[32P]-dATP

?-[32P]-dATP CAUTION! see Hint #2
3000 Ci/mmol ?-[32P]-dATP

10 mg/ml Bovine Serum Albumin

Oligo Labeling Buffer (5X) (See Hint #1) 250 mM Tris-HCl, pH 8.0
250 mM MgCl2
1 M HEPES, pH 6.6
100 ?M dTTP
100 ?M dGTP
3 ml of Random hexamers at an absorbance of 90 at 280 in TE buffer
100 ?M dATP

TE Buffer 10 mM Tris
pH 8.0
1 mM EDTA

Supplies

Tips
1. The 5X Oligo Labeling Buffer can be made in large batches and stored at -20?C for at least one year. For normal uses, it is only necessary to use one radioactive nucleotide. Store in small enough aliquot sizes that a single aliquot is thawed and refrozen for only one or two months. The buffer has the necessary cold nucleotides, so you need to make another one if you want to use two radioactive nucleotides in the labeling.

2. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.

3. Any quantity of DNA over about 5 ng works. Theoretically 20 to 40 ng gives the hottest probe. Over 100 ng of DNA will decrease the final specific activity.

4. A supercoiled plasmid may not label well. This can be remedied by a longer boiling step or, as a last resort, linearizing the plasmid before labeling.

5. If it's convenient, it is acceptable to incubate overnight.

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Labeling of DNA Probes using Primer