A. Preparation of the Y Oligomer: Kinasing Oligonucleotides, Annealing and Gel Purification
1. The standard substrate oligonucleotides are T1 (16-mer), T3 (30-mer), P1/P2L (84-mer), and P2L (70-mer). The T1 oligonucleotide is labeled by phosphorylation with γ-[32P]-ATP and T4 polynucleotide kinase and then annealed to P1/P2L, P2L and T3.
2. Set up the following kinase reaction mixture in a 0.5 ml microcentrifuge tube:
1 μl of 10X Oligo Kinase Buffer,
3 μl of γ-[32P]-ATP (approximately 0.5 mCi),
1 μl of 2 mg/ml Bovine Serum Albumin,
0.25 μl of T1 Oligonucleotide (1150 μg/ml, 50 pmol),
4 μl of ddH2O,
30 Units T4 Polynucleotide Kinase
3. Incubate at 37°C for 1 hr and then add 0.25 M EDTA.
4. Heat at 85°C for 15 min.
5. Add the following to the above reaction mixture: 20 μl P1/P2L oligonucleotide, 9.1 μl P2L oligonucleotide, 2 μl T3 oligonucleotide, 0.8 μl 5M NaCl, and 8.1 μl ddH2O.
6. Heat to 100°C for 3 min.
7. Put in a 80°C temperature block (or water bath) for 5 min and move the block (or water bath) to your bench and allow it to slowly cool to room temperature. Then transfer the tube(s) to ice.
8. Purify the Y oligomer by 10 % native gel purification. To make 40 ml of native gel mix, combine: 13.1 ml of 30 % Acrylamide:Bis Acrylamide (30:0.8),
1 ml of 20 X TBE buffer,
25.5 ml of ddH20,
0.2 ml of 10 % Ammonium Persulfate.
Then add 60 μl TEMED.
9. Add 40 μl of dye-sucrose mix to the Y oligomer sample and run the sample at 200 V until the Bromophenol Blue dye has migrated about two-thirds the way down the gel.
10. Autoradiograph and excise the slow-moving Y oligomer. The unlabeled T1 will migrate faster, as will the free γ-[32P]-ATP.
11. Excise the band and put it in a 1.5 ml microcentrifuge tube. Elute the DNA in 0.5 M Ammonium Acetate/10 mM Magnesium Acetate. Incubate the elution/gel mixture for 12 hours at 4°C on a shaking platform. Remove the liquid and repeat the elution 3 more times. Store each elution at 4°C.
12. Pool the four elution volumes in an Amicon Centricon-10 and store at 4°C during the other elutions.
13. Centrifuge the Centricon-10 at 9,700 X g (6,000 rpm using a Sorvall SS34 rotor) at 4°C to concentrate the sample.
14. Add 2 ml of ice-cold TEN to the concentrated sample and reconcentrate. Add an additional 2 ml of ice-cold TEN to the sample and reconcentrate to a final volume of 50-100 μl.
15. Invert the Centricon-10, centrifuge for 1 min at 9,700 X g (6,000 rpm using a Sorvall SS34 rotor) at 4°C and store the retentate at -20°C.
B. Disintegration Assay (both GTP and MgCl2 are essential cofactors for this reaction)
1. Use 2 μl of the Y oligomer per assay.
2. Set up the following reaction in 15 μl total volume: 2 μl Y oligomer, 1.5 μl 10 mM GTP, 1.5 μl 100 mM manganese chloride, 7 μl HGKED, 3 μl protein fraction of interest (in 0.1M KOH)
3. Incubate at 25°C or 30°C for 1 hr.
4. Add 15 μl deionized formamide dyes/30 mM EDTA.
5. Boil the samples and load 4 μl on a 15% urea polyacrylamide 0.4 mm thick sequencing gel using a clear 24-tooth comb. The Bromophenol Blue co-migrates with 10 nucleotide oligonucleotides on this percent gel. For a 40 ml solution to prepare a 15 % polyacrylamide urea gel, combine: 20 g Urea, 15 ml 40 % Acrylamide (19:1), 8.2 ml ddH2O, 0.4 ml 10 % Ammonium Persulfate (filter this mixture through a Whatman #1 filter), add 25 μl TEMED.
6. Transfer the wet gel to an film cassette and cover it with plastic wrap. Expose the gel to X-ray film at -80°C with an intensifying screen.
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