ChangBioscience.com
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
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DNA template (0.5-5 mg, less for single-stranded DNA)
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6 ml
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Sequencing primer (0.5 pmol/ml)
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2 ml
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5X Sequencing buffer
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2 ml
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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:
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DNA/primer mix (step 5)
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10 ml
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diluted labeling mix (1:5-1:15)
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2 ml
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100 mM DTT
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1 ml
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a-S35-dATP
(or a-P33-dATP,
or a-P32-dATP)
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1 ml
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diluted Sequenase (1:8)
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2 ml
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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.
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