DNA Reaction - Radiolabeling by Nick Translation


Jeff Lawrence

1. Prepare 10X Nick Translation Buffer:
	200 mM	Tris, pH 7.5	(200 mL	1 M Tris, pH 7.5)
	50 mM	MgCl2		(50 mL	1 M MgCl2)
	5 mM	DTT		(5 mL 	1 M DTT)
	500 mg/mL BSA		(250 mL 2 mg/mL BSA)
				(495 mL ddH2O)
2. Dilute DNase 1:5000 from 1 mg/mL stock.

3. Mix the following:
	2 µL 	DNA, about 100 ng
	1 µL 	10X Nick Translation Buffer
	1 µL 	1 mM dNTP (250 µM each)
	1 µL 	DNase dilution
	1 µL 	DNA Polymerase Holoenzyme
	4 µL 	α-32P-dATP
4. Incubate at 15° for 60 min.

5. Add 2 µL 250 mM EDTA, pH 8.0. Incubate at 65° for 5 min.

6. Run 3 mL Column Buffer through a G100 column. Column Buffer is:
	1.00 mM	Tris, pH 8.0	(100 mL 	1 M Tris, pH 8.0)
	0.25 mM	EDTA		(50 mL 	500 mM EDTA, pH 8.0)
				(100 mL	ddH2O)
7. Add 90 µL Column Buffer to reaction tube. Apply to G100 Column.

8. Monitor progress of radioactivity through the column with a geiger counter. When the radioactive front approaches the bottom of the column, collect 3 drop aliquots in microcentrifuge tubes. The labeled probe will run faster than the unincorporated radioactive nucleotide.

9. Allow remainder of solution, including unicorporated label, to run through the column.

10. Quantitate the aliquots in a scintillation counter and pool aliquots to yield a peak fraction of > 2x106 cpm.

11. Incubate peak fraction at 100° for 3 min to denature.

12. Place on ice. Apply to hybridization solution as necessary.


Last Update: Thursday, 19-Jun-2014 11:50:21 PDT
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Eric Kofoid eckofoid at ucdavis.edu