Random priming advice needed

Andrew Hobbs andrewh at uniwa.uwa.edu.au
Wed Dec 2 02:08:11 EST 1992


Hi, 

We normally use a slightly different procedure to that described in the
texts. This involves the following steps

1.	Make up the labelling reaction on ice with 32P-dCTP.

2.	Heat the DNA (in a volume of 10 ul) to be labelled to 100 C for
          five minutes.

3.	While boiling the DNA add klenow to the reaction tube, still on
          ice.

4.	Then add the DNA (somewhat less than 10 ul now due to
          evaporation) directly into the cold (0 C) incubation tube and
          mix immediately.   
         (DONT ADD MIXTURE TO DNA IN HOT (100 C) TUBE).

5.	Incubate at 37 C for 15 minutes.

I have used this with the Amersham kit although I normally make up my
own kit (1/4 the cost).  PEI cellulose chromatography indicates that
100 % of the 32P-dCTP is incorporated into DNA although Sephadex
columns (normal or spun columns) indicate that only 70 - 80% has been
incorporated into the added DNA.  The remainder I suspect is
incorporated into elongated hexanucleotides using other
oligonucleotides as templates.  If one uses this method then it is
important to purify using Sephadex.  Otherwise the filters get covered
with tiny hot spots (background) which I suspect are due to these hot
oligonucleotides.  
    The specific activity is determined solely by the amount of DNA
added and the amount of 32P-dCTP used.  We have achieved close to the
theoretical maximum specific activity of about 2-3 E9 cpm / ug. when
labelling 20 ng DNA with 50 uCi 32P-dCTP. (SA 3000 Ci/mM).

Hope that this is useful

Andrew Hobbs
Biochemistry
University of Western Australia

Andrewh at uniwa.uwa.edu.au



More information about the Methods mailing list