<p> Oligo Labelling of DNA Fragments (in agarose,too)</p><p>- 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. </p><p>- Slice the band out and trim to eliminate as much extraneous agarose as possible Weigh in an Eppendorf, and melt in 3 volumes dH O at 68o. 2</p><p>-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.</p><p>-While the DNA is boiling, you should mix the following in a 1.5 ml microtube:</p><p>10 µl OLB 2 µl 10mg/ml BSA 5 µl dCTP* (3000 Ci/mmol)</p><p>-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.</p><p>-Add 1 µl of Klenow fragment enzyme. Mix</p><p>-Incubate at least 2-3hrs at RT.</p><p>-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.</p><p>-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. </p><p>To make OLB:</p><p>100 µl 1M Tris, pH=7.5 250 µl 2M HEPES, pH=6.6 (pH w/ NaOH) 12.5 µl 1M MgCl 2 2.5 µl 50mM dATP 2.5 µl 50mM dGTP 2.5 µl 50mM dTTP 1.7 µl 2-mercaptoethanol 260 150 µl 90 A U/ml hexanucleotides (dissolve 50U in 550µl H O. 2</p>