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Re:purification of radioactive probe

Re:purification of radioactive probe - Protocols and Methods Forum

Re:purification of radioactive probe - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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Old 08-27-2007, 11:14 PM
Analia Alet
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Default Re:purification of radioactive probe



Hi Simone,

I used to make DNA probes for northern blot by using hexamers and the Klenow
fragment in the reaction mix. Anyway, after the reaction was finished, I
purified it with a Sephadex G-50 column. The reason I did so was because
according to Manniatis, Sephadex G-50 retains the unused labelled-nucleotides
and little oligonucleotides that may interfere in the blotting. After using
the column, the eluate was cheked for radiactivity, boiled 10 min, and
transferred to the hybridisation solution.

Once againg, please forgive me my english
Best luck
Analía

Lic. Analía I. Alet
Unidad de Biotecnología 1
IIB-INTECH
Camino Circunvalación Laguna km 6
Chascomús
Pcia de Buenos Aires
ARGENTINA
Tel: 0054 2241 430323 int102


Hi,

does anyone know if it is really necessary to purify a radioactive DNA probe
after labelling it by random priming? I want to use it on a northern blot.
So far, I worked with DIG-labelled probes, that do not need to be purified
after labelling. They are used directly in the hybridisation solution. Is it
different with radioactive probes? Do you think that precipitation by salt
and alcohol is sufficient for purification?

Thank you very much,
Simone

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