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Home  |  Protocols  |  Molecular Biology  |  Preparation of radioactive probes for nucleic acids blots

Protocol - Preparation of radioactive probes for nucleic acids blots

It generates highly specific and hot probes.

CategoryMolecular Biology
Creation dateJun 18, 2014
Last revisionJan 20, 2015
NameF. Anna Digilio
AddressInstitute of Biosciences and BioResources - CNR, Via P. Castellino 111, 80131 Naples, Italy
Phone+39 081 6132430


Description Temperature Time Note
Prepare DNA template (300-600 bp) by PCR/restriction + gel purification
Prepare a normal PCR reaction with the final concentrations ot the radionuclide as reported below
Parameters: Reaction volume: 25 ul; Taq: e.g. Roche, 1U for 25 μl rxn; DNA: 2 ng/ul rxn; Primer: 1 μM each
Start a PCR cycle as reported
No need for denaturing
You can also use just one primer in order to obtain a probe complementary to the mRNA
the primer should be internal to that used for preparing the template through PCR.

Other informations

Radionuclide final concentrations:


dAGTTP* uM each]

Cold dCTP [uM]

Hot dCTP [uM]

ul of standard (3.33 uM) hot dCTP in 25ul

Low [2:1]





High [1:1]





a 1:1 for sensitive applications.

* also 100 uM worked.


Standard radionuclide:

32P, alpha position, dCTP

10 uCi/ul

3000 Ci/mmol (= 3.33 uM)

colored buffer

  • Cycling:
    • 1x        94°C     1’
    • 40-60x (usually 50x)
      • 94°C     20’’
      • An.T   30’’
      • 72°C     1’30’’-2’ (6’40’’/kb or 0.4’’/nt with 100 uM dAGT-tp and Taq)


Quality validation: Yes

Validation info

The protocol has been used to obtain data published  in a peer-review journal.


Cavaliere D, Di Cara F, Polito LC, Digilio FA. Cloning and functional characterization of the intersex homologous gene in the pest lepidopteron Maruca vitrata. Int. J. Dev. Biol. 2009;53:1057-1062


Funded byGrant from the Regione Campania (L.R. 31.12.1994 n.5)


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