preparation of probes for northern blotting - preparation of probes for northern blotting (May/03/2006 )
A hot good evening to all of you.
this is again a very pleasure of mine to have in communication to all you mentors.
this time i m passing a different problem. i want to check the expression level of mrna for my gene
. i have developed the primers and want to do Reverse transcriptase PCR. but i came to know that
this is only qualitative. Now i want to see the expresion level of mRNA by northern blotting. and
want to lable my primer( 20 base length).
now i have query that will 20 base length primers are sufficient for the preparation of probes. and
i will do the endlabelling of primers. i want ot do it conventionally ( not by kits).
thanx in advance.
oligo of 20bases are sufficent for designing probe as i know. I use to label them by T4polynucleotide kinase and gamma P32 ATP.
10pmol of probe in 50µl reaction volume, purify with a nucleotide removal kit or similar.
incubation in church buffer.