TheE.Z.N.A.®MagBind™DyeterminatorRemovalProcedure

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).