Mag-Bind RX Plasmid Isolation Protocol

一、实验试剂  Absolute (96%-100%) ethanol   二、实验设备 1.  Centrifuge with swinging-bucket rotor at room temperature capable of 3000 x g (such as Eppendorf 5810 with MTP rotor) 2.  Adapter for 96-well deep-well plate 3.  Magnetic Separation Device (Cat# MSD-01 & MSD-01B) 4.  96 well Process Plate (Cat# EZ9604) 5.  Vacuum oven or incubator preset to 70℃ 6.  Multiple Channel Pipettor   三、实验步骤 1.  Culture Volume: Innoculate 1.0-1.5 ml LB/antibiotic(s) medium placed in a 96-well 2 ml culture plate and grow at 37°C with agitation fo plate/block with E.coli for 12-16 h. 2.  Seal the plate with tape or film and pellet bacteria by centrifugation at 3000 x g in a swinging-bucket rotor at room temperature for15minutes at room temperature. 3.  Remove the tape and discard supernatant into a waste container. Dry the plate by placing upside-down on a paper towel to remove excess media. 4.  Add 90 ul Solution I/RNase A and 30ul Mag-Bind Particle LC to the bacterial pellet in each well of the deep well plate. Resuspend cells completely by shaking or pipetting. Complete resuspension of cell pellet is vital for obtaining good plasmid yields. 5.  Add 120 ul Solution II and mix by gently shaking and rotating the plate for 1 minute to obtain a cleared lysate. A 5 min incubation at room temperature may be necessary. Avoid vigorous mixing as doing so will shear chromosomal DNA and lower plasmid purity. (Store Solution II tightly capped when not in use.) 6.  Add 120 ul Neutralization Buffer and mix by gently shaking and rotating the plate for 1 minute until precipitate forms. 7. Transfer the lysate into a new Process Plate (Cat# EZ9604) and place the plate on the magnetic separation device (Cat# MSD-01). Allow the lysate to clear. A tight pellet should form in the corner of each well, which allows the a pipette tip to descend to the center of the well without disturbing the pellet. 8.  Transfer 200 ul of cleared cell lysate into another new Process plate. 9.  Add 10 ul Mag-Bind Particles SX followed by 70 ul PFC Buffer. Mix the sample throughly by pipetting up and down for 10 times. 10. Place the plate onto the magnetic separation stand to magnetite the magnetic particles. Remove the supernatant after the magnetic particles have completely migrated to the walls of each well adjacent to the magnets. (Supernatant should be clear when migration is complete.) 11.  Remove the plate from the magnetic separation stand, then wash the pelleted Mag-Bind particles by adding 200ul SPM Wash Buffer. Resuspend the particles by pipetting up and down for 20 times. Again place the plate on the magnet separation stand and remove the supernatant after Mag-Bind particles have completely migrated to the walls of the plate. NOTE: For better washing efficiency, Mag-Bind particles should be fully resuspended. 12.  Remove the plate from magnetic separation stand and wash the Mag-Bind particles by adding another 200 ul SPM Wash Buffer to each well. Wash the Mag-Bind particles by pipetting up and down for 10 times . Place the plate on the magnetic separation stand to magnetize the Mag-Binds particles. Aspirate the supernatant. 13.  Optional: Remove the plate from magnetic separation stand and wash the Mag-Binds particles by adding 200 ul absolute ethanol to each well. Resuspend the Mag-Binds? particles by pipetting. Place the plate on the magnetic separation stand to pellet the Mag-Binds particles. Aspirate the supernatant. 14.  Air dry the Mag-Binds particles pellet for 5-10 minutes at room temperature. 15. Elute DNA: Resuspend the Mag-Bind? particles pellet with 50-100ul water or TE buffer by pipetting up and down for 40 times. 16.  Place the plate onto the magnetic separation stand to pellet the Mag- Binds? particles. 17.  Transfer the supernatant containing the purified plasmid into a clean 96-well microplate.