Single-stranded dye-terminator reactions required approximately 2 ug of phenol extracted M13-based template DNA. The DNA is denatured and the primer annealed by incubating DNA, primer, and buffer at 65degC. After the reaction cooled to room temperature, alpha-thio-deoxynucleotides, fluorescent-labeled dye-terminators, and diluted Sequenase[TM] DNA polymerase are added and the mixture is incubated at 37degC. The reaction is stopped by adding ammonium acetate and ethanol, and the DNA fragments are precipitated and dried. To aid in the removal of unincorporated dye-terminators, the DNA pellet is rinsed twice with ethanol. The dried sequencing reactions could be stored up to several days at -20degC.

Double-stranded dye-terminator reactions required approximately 5 ug of diatomaceous earth modified-alkaline lysis midi-prep purified plasmid DNA. The double-stranded DNA is denatured by incubating the DNA in sodium hydroxide at 65degC, and after incubation, primer is added and the reaction is neutralized by adding an acid-buffer. Reaction buffer, alpha-thio-deoxynucleotides, fluorescent-labeled dye-terminators, and diluted Sequenase[TM] DNA polymerase then are added and the reaction is incubated at 37degC. Ammonium acetate is added to stop the reaction and the DNA fragments similarly are precipitated, rinsed, dried, and stored.

Protocol

For Single-stranded reactions:

1. Add the following to a 1.5 ml microcentrifuge tube:

4ul ssDNA (2ug)

4ul 0.8uM primer

2ul 10x MOPS buffer

2ul 10x Mn[2+]/isocitrate buffer

12ul

2. To denature the DNA and anneal the primer, incubate the reaction at 65-70deg C for 5 minutes. Allow the reaction to cool at room temperature for 15 minutes, and then briefly centrifuge to reclaim condensation.

3. To each reaction, add the following reagents and incubate for 10 minutes at 37degC. (For more than one reaction, a pot of the reagents should be made).

7ul ABI terminator mix (401489)

2ul diluted Sequenase[TM] (3.25U/ul)

1ul 2 mM a-S dNTPs

22ul

The undiluted Sequenase[TM] (70775) from United States Biochemicals is 13 U/ul and should be diluted 1:4 with USB dilution buffer prior to use resulting in a working dilution of 3.25 U/ul.

4. Add 20 ul 9.5 M ammonium acetate and 100 ul 95% ethanol to stop the reaction and vortex.

5. Precipitate the DNA in an ice-water bath for 10 minutes. Centrifuge for 15 minutes at 12,000 rpm in a microcentrifuge at 4degC. Carefully decant the supernatant, and rinse the pellet by adding 300 ul of 70-80% ethanol. Vortex and centrifuge again for 15 minutes, and carefully decant the supernatant.

6. Repeat the rinse step to insure efficient removal of the unincorporated terminators. (Alternatively, after the first rinse step, droplets of supernatant can be removed by carefully absorbing them with a Q-tip cotton swab or a rolled up Kimwipe).

7. Dry the DNA for 5-10 minutes (or until dry) in the Speedy-Vac, and store the dried reactions at -20degC.

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