The E.Z.N.A.® Mag-Bind™ Dye terminator Removal Procedure

实验概要

Excess  unincorporated, nonradioactive label can cause high background  fluorescence in automated sequencing gels. For optimal sequencing  results, remaining labeled dideoxynucleotides should be removed prior to  electrophoresis. The E.Z.N.A.^® Mag-Bind™ Dye-Removal Kit is  designed for effective and reliable removal of unincorporated  terminators from sequencing reactions

Magnetic particles offer greater flexibility than centrifugation-and  vacuum-based formats for nucleic acid purification. Among these  benefits are scalability, easier handling and in-solution kinetics. The  purification process results in highly purified sequencing products.

主要试剂

1. Absolute (96%-100%) ethanol

2. Molecular biology grade water

主要设备

1. Multiple-channel pipettor

2. 96-well microplate

3. Magnetic Separation Devices

实验步骤

1. Determine the  volume of the crude sequencing product and transfer the sample into  96-well microplate. Add 10 ul molecular grade water to each sample.

2. Add 2 volume of Mag-Bind^® Particle/ethanol mixture and mix thoroughly by pipetting.

3. Incubate 10 minutes at room temperature. Place the microplate on a  magnetic separation device designed for 96-well plate. Wait for 5  minute or until the solution is clear of beads.

4. Aspirate entire solution with multiple channel pipettor. Do not disturb the Mag-Bind™ Particle pellet.

5. Dispense 100ul MPG Wash Buffer into each well, resuspend Mag-Bind  Particle pellet by pipetting. Incubate for 1 minute at room temperature.

6. Place the microplate on a magnetic separation device, and wait 1 minute or until the solution is clear of beads.

7. Aspirate entire solution with a multiple channel pipettor. Do not disturb the Mag-Bind™ Particle pellet.

8. Dry the Mag-Bind™Particle pellet by air for 10 minutes.

9. Remove the plate from separation device. Add 15ul - 30ul water to each sample and resuspend Mag-Bind^® Particle pellet by pipetting.

10. Incubate 10 minutes at room temperature. Place the microplate on a  magnetic separation device designed for 96-well plate. Wait for 1  minute or until the solution is clear of beads.

11. Transfer the cleared supernatant to a new microplate (not provided).