For Double-stranded reactions: Sequenase[TM] catalyzed seque

Thursday, November 20, 2003

Description
For Double-stranded reactions

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

5 ul ds DNA (5 ug)
4 ul 1 N NaOH
3 ul ddH2O
12 ul

2. Incubate the reaction at 65-70degC for 5 minutes, and then briefly centrifuge to reclaim condensation.
3. Add the following reagents to each reaction, vortex, and briefly centrifuge:

3 ul 8 uM primer
9 ul ddH2O
4 ul MOPS-Acid buffer
28 ul

4. 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).
4 ul 10X Mn[2+]/isocitrate buffer
6 ul ABI terminator mix
2 ul diluted Sequenase[TM] (3.25 U/ul)
1 ul 2 mM [alpha]-S-dNTPs
22 ul

The undiluted Sequenase[TM] 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.
5. Add 60 ul 8 M ammonium acetate and 300 ul 95% ethanol to stop the reaction and vortex.

6. 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 80% ethanol. Vortex and centrifuge again for 15 minutes, and carefully decant the supernatant.

7. 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 Kim-wipe).

8. Dry the DNA for 5-10 minutes (or until dry) in the Speedy-Vac.

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For Double-stranded reactions: Sequenase[TM] catalyzed seque