SEQUENCING USING SEQUENASE |
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Last Update: December 2006 |
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| PREPARE SOLUTIONS | |
| 1. Dilute Sequenase enzyme: | Dilute 1:8 ratio in dilution buffer (or TE) |
| 2. Dilute Labelling nucleotide mix: | Dilute 1:5 ratio in water |
| 3. Sequencing gel: | Mix 30.2 g of Urea, 17.2 mL of 10X TBE, 7.2 mL of 50% Acrylimide (Long ranger), and 31.3 mL of dH2O. 5. Stir into solution (warm). Filter through Buchnner funnel (total: 72 mL). Add 300 mL of 10% APS and 35 mL of TEMED right before loading. |
| 4. Pour sequencing gel: | Hit plates as gel is poured for even distribution and preventing air bubbles. Level gel solution with plates for an adequate rate. Check tubing before pouring. Place any visible glass chips towards to top, outer part of the gel. Let gel polymerize overnight if possible. Check for any leaks |
| 5. 5X TBE (1 L): | Mix 54 g of Tris base, 27.5 g of boric acid, 20 mL of 0. M EDTA, pH 8.0, and dH2O to 1 L
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| 6. 0.5X TBE (2 L): | Mix 200 mL of 5X TBE with 1,800 mL of dH2O |
| PROCEDURE | |
| 1. Set up Labelling reaction: 1 mL of 0.1M DTT, 2 mL of diluted labelling nucleotide mix, 0.5 mL of 35S-dATP (5mCi), and 2 mL of diluted sequenase enzyme (total volume of 15 mL [10 mL of DNA + 5 mL of label]) | od|
| 2. Incubate for 5 minutes at room temperature | |
| 3. Label 4 tubes G,C,T,A; fill each with 2.5 mL of termination mixture (ddNTPs). Pre-warm tubes to 65oC-72oC | |
4. Transfer 3.5 mL of labelled reaction to tubes with termination mix (G, C, T, and A) |
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* Do 5 reactions at a time, timing each reaction (step A) at 1 minute intervals so that at the end of the 5th reaction, the first one is ready for this step (step B). Do all four nucletide termination reaction in 1 minute to continue with the next step A reaction on time |
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5. 5 minute incubation. Add 4 mL of stop solution to each termination reaction. Store on ice |
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| * Time each stop (5) at 1 minute intervals as in the previous step | |
6. LOAD gel: heat samples to 75oC-80oC for 5 minutes. Stop by placing reactions on ice. Load 2-3 mL of DNA in each lane |
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7. Prepare sequencing gel |
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| 8. Place gel in gel apparatus and pre-warm (45oC-50oC) (pre-warm for about 45 minutes) | |
| 9. Load samples | |
| 10. Run at 1500 V and 75 mA | |
11. Take gel out of glass plates, place on 3MM paper, and dry |
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12. Expose gel to film. Develop film. Read sequence |
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