A good Oligolabelling protocol*****
smori at nmsu.edu
Sun Jun 4 21:54:49 EST 1995
Colin Rasmussen (crasmussen at anat.med.ualberta.ca) wrote:
: We use the procedure outlined in the newer editions of the Maniatis
: cloning manual. The procedure is based on the original work of Feinberg &
: Vogelstein and works very well in our hands. We get our random hexamers
: from Pharmacia, use Promega Klenow, and Amersham 32P-dCTP. We typically
: use about 50ng DNA (purified on LMP agarose) in a 25ul reaction incubated
: at 37°C for 1-2 hrs. The probe is separated from the unincorporated dNTPs
: on G-50 minicolumns and we usually get 60-80% incorporation per 50uCi 32P
: In regards to helping you with your problem, what do you mean by less than
: acceptable. Many factors can affect the efficiency of labelling and it
: would be useful for you to be more specific.
Dear Colin, and all who have responded;
I have been getting incorporations less than 10 fold in my
oligo-labelling reactions. I have been following FOCUS protocol which
asks for 3ug of random hexamers. What I am trying to oligo-label is PCR
fragments from DDRT-PCR which have been NaOAC ppted. The FOCUS protocol
asks for 40 Units of Klenow which I think is so rediculously high that, I
have lowered it by 10 fold. I incubate my reaction at 25C for 3 hrs. I
hope this is long enough. I use 5ul of 3000Ci/mmole 32PdCTP in my
reaction mixture. The concentration of other dNTP's are 0.2 umoles. The
reaction mixture's volume is 50ul.
I have done it 3 times and all three times I have been getting inadequate
I have been tempted to forget this and just do an end-labelling reaction.
What do you think?
C A N A D A
Program in Molecular Biology
Department of Chemistry and Biochemistry Box 3C
New Mexico State University
Las Cruces NM
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