dsDNA sequencing
A. Double stranded DNA denaturation:
1. Combine in a 1.5 ml eppendorf tube:
8 ul of miniprep plasmid DNA
2 ul of 2M NaOH
2. Incubate at 65oC for 10 min.
3. Add:
7 ul Water
3 ul 3M Acetate
60 ul Ethanol
4. Incubate at -20 oC overnight.
5. Wash the pellet once with 500 ul of cold (-20 oC) 70% (v/v) ethanol
6. Dry the pellet (almost invisible) 10 min in a speedvac
7. Go to annealing.
B. ANNEALING:
1. Add to denatured and dried plasmid DNA
PRIMER -40 PRIMER RSP
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6 ul Water 6ul Water
2 ul of 5x Sequenase Buffer 2 ul of 5x Sequenase Buffer
2 ul of primer (0,5 pmol/ul) 2 ul of primer (0,5 pmol/ul)
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2. Heat to 65oC for 2 min, and slowly cool till room temperature
(>30 min) till temperature is <30 oC. These are the ANNEALING TUBES.
3. Label 8 0.5ml Eppendorf tubes: R R R R U U U U
A C G T A C G T
4. Aliquote 2.5 ul of each solution of ddA, ddC, ddG e ddT (Sequenase
Kit) in each of the tubes of item [3].
C. SEQUENCING:
1. "Spin Down" the contents of the ANNEALING TUBES.
2. Ad to the ANNEALING TUBES, in order:
1 ul of 0,1 M DTT
2 ul of labelling mix (1x)(OBS.: DILUTE THE 5X STOCK BEFORE USING)
0,5 ul of a35S-dATP (>1000 Ci/mmol; >10 uCi/ul)
3. Dilute Sequenase:
for 3 tubes for 6 tubes
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SEQUENASE 0,83 ul 1,65 ul
Dilution Buffer 5,81 ul 11,55 ul
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4. At intervals of 1.5 min ad 2 ul of diluted SEQUENASE to the
ANNEALING TUBES. Pump to mix (DO NOT MAKE BUBBLES!)
6. Let them stand at room temperature for 13 minutes.
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|ONE MINUTE BEFORE THE TIME (i.e. 12 MIN AFTER THE BEGINNING OF THE REACTION |
|PUT THE 4 TUBES [A,C,G,T] at 37 oC |
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7. After that, add 3.5 ul of labelling mix to each of the [A,C,G,T]
tubes and let them stand at 37 oC for 5 min.
8. After 5 min add 4 ul de stop mix to each of the [A,C,G,T] tubes.
9. Store at -20 oC.
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TIME TABLE (min)
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SEQUENASE TRANSFER TO [A,C,G,T] STOP MIX
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1:30 14:30 19:30
3:00 16:00 21:00
4:30 17:30 22:30
6:00 19:00 24:00
7:30 20:30 25:30
9:00 22:00 27:00
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