DNA序列分析（DNA sequencing)-试验方案

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DNA序列分析（DNA sequencing)
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4.0 µL

Thermo Sequenase I DNA polymerase (10 U/ul)

1.0 µL

Distilled Water (to a final volume of 27 µL)

____ µL

Total volume

27.0 µL

3.Flick the tubes gently to mix—do not vortex. Centrifuge the tubes briefly (10 sec. at max rpm) and return to ice immediately.
C. Bringing the termination mixes and DNA Master Mixes together (Estimated Time 15-30 minutes)
4. Label four 0.2 mL PCR tubes for each DNA Master Mix “A, C, T, and G”.

9.Aliquot 6 µL of the Master Mix from section B into each of the 0.2 mL PCR tubes you have just labeled, A, C, T, and G. Return tubes to ice.
10.Aliquot 2 µL of the corresponding termination mix ddA, ddC, ddG, ddT from section A into each A, C, T, G labeled 0.2 ml PCR tube. Return tubes to ice

11.Flick the tubes gently to mix—do not vortex. Centrifuge the tubes briefly (10 sec. at max rpm) to collect at the bottom of the tube and return to ice immediately.
D. Programming the Thermalcycler (Estimated Time 15-25 minutes for set up and 2.5 -3.0 hours for cycling)
12.Program and run the thermocycler (PCR machine) using the following parameters.

**It is a good idea to test the program parameters before beginning the run.
E. Preparation for Sequencing
NOTE: Unincorporated dye terminators (ddNTPs) can be removed from the sequencing reactions by ethanol precipitation of the DNA.
13.Centrifuge the tubes briefly (10 sec. at max rpm) to collect at the bottom of the tubes.
14.Transfer the PCR reactions into labeled 1.5 microfuge tubes. *There will be one 1.5 mL microfuge tube for every PCR tube. Be careful and descriptive with labeling and transferring.
15.To each labeled 1.5 mL microfuge tube add:

2µL of 7.5mM Ammonium acetate (provided by TA’s)
2µL Glycogen solution (provided with Sequencing Kit)
30µL (3x reaction volume) of chilled 100% ethanol (provided by TA’s)

Mix thoroughly by flicking the tubes, centrifuge the tubes briefly (10 sec. at max rpm) to collect at the bottom of the tubes and place in -20°C for 20-45 minutes to precipitate the DNA.
16.Centrifuge all tubes at full speed (room temperature) in a table-top microcentrifuge for 20-30min to pellet the precipitated DNA.
17. Carefully remove all supernatant with a 10 µL pipet . (A large tip can cause damage to your DNA). All ethanol must be removed from the tube!
18. Carefully add 200 µL of chilled 70% ethanol to the tube and briefly flick the tube to resuspend the pellet. (Pellets are very fragile at this point and are clear—may be hard to see.) Centrifuge at full speed for 5 minutes. This provides and additional wash.
19. Carefully remove the supernatants with 10 µL pipet and air dry the pellet on the table top for 5 minutes by leaving the lid on the tube open. If pellets are overdried, they may be difficult to resuspend.
20.Resuspend each pellet in 6 µL of Formamide (provided with Sequencing Kit) loading dye and vortex vigorously (10-20 sec). Briefly centrifuge to collect sample at the bottom of the tube. Return samples to ice.
*The DNA pellets must be completely resuspended at this step for optimal sequencing results.
F. Sequencing
The ALFExpress equipment will be set up by the instructor or TA.
21.Just prior to loading the samples onto the gel, heat each sample to 70ºC in a water bath for 2-4 min to denature, and then cool immediately on ice.
22.Using the 100 uL flat tips, load the entire volume (6µL) of each sample into separate lanes of the sequencing gel.

After all samples have been loading the sequencing process will be initiated by the instructor or TA.

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