Automated fluorescence sequencing using ABIPRISMRigDye end chemistry is one of the methods for efficient DN eight sequence determination. Sequence gradients are generated by the standard Sanger method using fluorescent labeling of nucleosides at the end of the chain. This experiment was derived from PCR Laboratory Guide (Second Edition) by Seed Kang and Qu Lijia.

Operation method

Clean purification experiments for stained ends

Materials and Instruments

Ethanol Elution Sample Solution
Magnetic separation device Plate holder Plate rack Sequencing reaction purification system

Move

I. Materials

1. Buffers, solutions and reagents

Gel elution/sampling solution for sequencing (see Table 9-4)

90% Ethanol Wash

2. Specialized equipment

MagnaBot II Magnetic Separator (Promega)

Plate Clamp 96 (Promega)

Plate Rack (Promega)

3. Other

WizardMagneSil Sequencing Reaction Purification System (Promega; includes MagneSilGREEN [paramagnetic particles])

96-well plate

II. Methods



1. Load the 96-well plate into the Plate Clamp 96 [Figure 9-10(a), (b)].

2. Place the plate on the automated mechanical bench using the plate rack [Figure 9-10(c)].

3. Vigorously shake the vial to resuspend the MagneSil pellets, adding 180ul of MagneSil pellets per 20ul of Sequencing Reaction System.

4. incubate at room temperature for 5 min, during which time aspirate mixing is performed at 0, 2.5 min, and 5 min, respectively.

5. Place the plate on the MagnaBot II Magnetic Separation Device [Figures 9-10(d)] to capture the particles.

6. Remove the liquid to avoid losing any particles.

7. Remove the plate from the MagnaBot II device and place it on the plate rack.

8. Add 100ul of 90% ethanol to each sample.

9. Incubate at room temperature for 5 min, during which time mixing is performed by aspiration at 0, 2.5 min and 5 min, respectively.

10. Place the plate on a MagnaBotn II magnetic separation device to capture the particles.

11. Remove the liquid to avoid loss of any particles.

12. Repeat steps 7~11 to bring the total number of washes to 2.

13. Air dry the particles at room temperature for about 10 min.

14. Add appropriate amount of elution/sampling solution (Table 9-4).

15. Incubate at room temperature for 1~2 min.

16. Place the plate on the MagnaBotn II Magnetic Separation Device to capture particles.

17. Transfer the purified sequencing reaction system to a clean 96-well plate, being careful to avoid losing any particles.

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