Oligo Labelling of DNA Fragments (in agarose,too)
- The DNA fragment to be labelled should either have been purified (in which
case, use 100ng for labelling) or should be run on a low melting point
agarose gel (TBE) made with Millipore water in a clean box.
- Slice the band out and trim to eliminate as much extraneous agarose as
possible Weigh in an Eppendorf, and melt in 3 volumes dH2O at 68o.
-Put a total of 35µl of the melted DNA fragment (or melted fragment + water if
less DNA will be sufficient; or pure fragment + water) in a screw-cap
microtube and boil for 3 min. Store at 37o until ready to add to reaction.
-While the DNA is boiling, you should mix the following in a 1.5 ml microtube:
10 µl OLB
2 µl 10mg/ml BSA
5 µl dCTP* (3000 Ci/mmol)
-Add 32µl of the boiled, cooled DNA (a fast way of cooling it is to slowly drip the
DNA down the side of the tube) to the above mixture.
-Add 1 µl of Klenow fragment enzyme. Mix
-Incubate at least 2-3hrs at RT.
-Add 150µl TE, heat at 65° to melt, and count in probe counter. Run thru a
G-50 spin column to remove unicorporated nucleotides. Put into 1.5 ml
microtube and re-count. Calculate % incorporation. If less than 25%,
throw away. 25-50% : use for one genomic blot. 50-75 % - good for 2
blots. > 75% - good for three blots.
-Boil desired amount of probe 3 - 5' in a screw-top microtube, afterward
keeping on ice until adding to the hybridization. Remainder of probe can
be stored up to 1.5 weeks at -20o.
To make OLB:
100 µl 1M Tris, pH=7.5
250 µl 2M HEPES, pH=6.6 (pH w/ NaOH)
12.5 µl 1M MgCl2
2.5 µl 50mM dATP
2.5 µl 50mM dGTP
2.5 µl 50mM dTTP
1.7 µl 2-mercaptoethanol
150 µl 90 A260U/ml hexanucleotides
(dissolve 50U in 550µl H2O.