Ambion, Inc. - Determining DNA Probe Specific Activity

Technical Resources > Appendix > Tools & Calculators

DNA Probe Specific Activity Calculator

This interactive calculator will walk you through the calculations to determine the specific activity of a radiolabeled DNA probe produced by random priming.

Step 1. Calculate Stock Concentration of Radiolabeled Nucleotide

What is the specific activity of your radiolabeled nucleotide stock (Ci/mmol)?

What is the concentration of your radiolabeled nucleotide stock (mCi/ml)?

The µM concentration of your radiolabeled nucleotide stock is: µM

Radionuclide concentration (mCi/ml)		Specific activity of radiolabeled nucleotide (Ci/mmol)		Ci		1000 ml		1000 µmol		µ M radiolabeled nucleotide
	x		x		x		x		=
				1000 mCi		L		mmol

Step 2. Calculate the Moles of Labeled Nucleotide in Your Labeling Reaction

What is the volume of radioactive nucleotide
in your labeling reaction (µl)?

The moles of labeled nucleotide in the reaction is: pmol

Vol radiolabeled nucleotide (µl)		Conc radiolabeled nucleotide (µM)		1 L		mol		10¹² pmol
	x		x		x		x		=	pmol nucleotide
				10⁶ µl		10⁶ µmol		mol

Step 3. Determine the Efficiency of Radiolabeled Nucleotide Incorporation

To perform this step, you must have counted two aliquots of reaction product: one counted after addition of the radiolabeled nucleotide before any other manipulation, and the other counted after removal of unincorporated nucleotides.

Unincorporated nucleotides can be removed by TCA precipitation or spin column (see Technical Bulletin 174 for details on TCA precipitation). Because spin columns dilute your sample, the calculations are slightly different depending on the method of unincorporated nucleotide removal. Use only one (A or B) of the below sets of calculations.

A. TCA Precipitation Method
(two aliquots are removed after the reaction: the first is counted directly and the second is TCA precipitated and then counted)

What was the final reaction volume just prior to aliquot removal (µl)?

What was the volume of the aliquot counted
with no other manipulation (µl)?

How many counts per minute were found for the aliquot counted
with no other manipulation (cpm)?

What was the volume of the aliquot from which unincorporated nucleotides were removed prior to counting (µl)?

How many counts per minute were found for the aliquot from which unincorporated nucleotides were removed (cpm)?

The percent of radiolabeled nucleotide incorporated is %

cpm/µl after removal of unincorporated nucleotides		Dilution factor		100%		percent incorporation
	x		x		=
cpm/µl before removal of unincorporated nucleotides

B. Spin Column Method
(an aliquot is removed directly after the labeling reaction; the remaining reaction product is put through a spin column)

What was the final reaction volume before spin column treatment (µl)?

What was the volume of the aliquot removed (before spin column treatment) (µl)?

How many counts per minute were found for the aliquot taken prior to spin column treatment (cpm)?

What was the total volume of the reaction after spin column treatment (µl)?

What was the volume of the aliquot removed after spin column treatment (µl)?

How many counts per minute were found for the aliquot taken after spin column treatment (cpm)?

The percent of radiolabeled nucleotide incorporated is %

cpm/µl after removal of unincorporated nucleotides		Dilution factor		100%		percent incorporation
	x		x		=
cpm/µl before removal of unincorporated nucleotides

Step 4. Calculate the Moles of Labeled Nucleotide Incorporated into Product DNA

The moles of labeled nucleotide incorporated is: pmol

percent incorporation

µl radiolabeled nucleotide added

µM radiolabeled nucleotide

mol

10¹² pmol

pmol nucleotide incorporated

100%

10⁶ µl

10⁶ µmol

mol

Step 5. Calculate the Amount of DNA Synthesized

The amount of DNA that was synthesized is ng

pmol nucleotide incorporated		330 g		4 (assumes equimolar amt of all 4 nt)		mol		10⁹ ng		ng DNA synthesized
	x		x		x		x		=
		mol				10¹² pmol		g

Step 6. Calculate the Total Amount of DNA

How much tempate DNA was used in the reaction (ng)?

The total amount of DNA is ng

ng template DNA

ng DNA synthesized

ng total DNA

Step 7. Determine the Counts per Minute Incorporated into Product

The cpm incorporated into the probe is cpm

percent incorporation

100%

Vol Radiolabeled Nucleotide (µl)

Conc Radiolabeled Nuc (mCi/ml)

2.222 x 10⁶ dpm

µCi

1000 µl

1000 µCi

mCi

cpm incorporated

Step 8. Calculate the Specific Activity of the Probe

The specific activity of the probe is cpm/µg

cpm incorporated

ng total DNA

1000 ng

µg

specific activity (cpm/µg)

Note: These calculations assume complete removal of unincorporated nucleotides and 100% recovery from spin column treatment or TCA precipitation.