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Star Republic: Guide for Biologists

Sequencing (Dideoxy Chain Termination)
The following protocol uses Sequenase Kit from USB.

1. Mix 18 ml plasmid DNA and 2 ml of 2 M NaOH/2mM EDTA. Incubate at room temperature for 20 minutes. This denatures double-stranded DNA. If the template is single-stranded go to step 5.

2. Add 2 ml ammonium acetate (pH 4.5) and then 50 ml 100% ethanol. Precipitate on ice for 15 minutes.

3. Centrifuge at maximum speed in a microfuge at 4 C for 15 minutes and carefully discard the supernatant.

4. Wash the pellet with 70% ethanol, decant the supernatant. Air dry. Resuspend the pellet in 6 ml of ddH2O.

5. Mix

DNA template (0.5-5 mg, less for single-stranded DNA) 6 ml
Sequencing primer (0.5 pmol/ml) 2 ml
5X Sequencing buffer 2 ml

6. Heat to 65 C for 2 minutes and allow to cool down slowly to room temperature over 30 minutes.

7. Dilute labeling mix 1:5 to 1:15, higher dilution for sequencing shorter distances from the primer. Dilute Sequenase 1:8 in ice-cold TE immediately before use. Add to the tube:

DNA/primer mix (step 5) 10 ml
diluted labeling mix (1:5-1:15) 2 ml
100 mM DTT 1 ml
a-S35-dATP (or a-P33-dATP, or a-P32-dATP) 1 ml
diluted Sequenase (1:8) 2 ml

Allow reaction to extend at room temperature for 3-5 minutes.

8. Add 2.5 ml termination mix (ddGTP, ddATP, ddTTP, ddCTP) into separate prelabeled tubes or separate wells in 96-well plate. Warm to 37 C.

9. Dispense 3.5 ml of the reaction mixture (step 7) into each of the termination mix and incubate at 37 C for 5 minutes.

10. Add 4 ml Sequenase stop solution to each tube/well. Seal the tubes/wells. Store at -20 C.

11. Heat samples to 80-90 C for 2 minutes, put on ice and immediately load 1-3 ml per lane.