purification of radioactive probe

Analia Alet via methods%40net.bio.net (by analia_alet from intech.gov.ar)
Mon Aug 27 18:14:54 EST 2007


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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