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hnRNA isolation (from fresh blood)
Posted by: schopper (IP Hidden, New member, 4)
Date: June 6, 2006 09:58AM
hallo,
does anyone know a nice protocoll for isolation of nuclear RNA, beside the one from Wilkinson (1988) which is given here in the method section? the method i use is based on the rna methologies book from robert e. farrell, jr. // a comibination of cellular lysis to get the nucleus and subequent isolation of the nuclear RNA, but what i get is a DNA band but not the characteristic 28S and 18s band, which looks to me that i isolated somehow DNA but no RNA, which makes me very sad :-( in one step of the protocol it is recommend to use DNase I (RNase-free) in a concentration of 50 U/ ml !!! I use the DNase from quiagen, that is provided with kunitz, but i use this kunitz like normal units! Can this be problematic??? Another thing is that i should use phenol saturated with NETS buffer (10 mM Tris; pH 7,4 100mM NaCl; 10mM EDTA; 0,2 % SDS Is essential for isolation of RNA to saturate, cause i did not do it? hope someone can help me, i am at the very beginning of my science expirience as u can see!!!! thanks schopper
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