DNA extraction v9.0 (modified BOMB)
Yin-Tse Huang, Tsu-Chun Hung, Guan Jie Phang
Abstract
DNA extraction using yttria-stabilized zirconia beads lysing and automated magnetic bead-based extraction.
Steps
DNA purification
Add 100µL of magnetic beads (10 mg/ml) to the 1st well of 96 deep well plate
Sample Collection
Add 200µL of 0.5 mm beads to a 2mL screw tube.
Add 200µL of 1 mm beads to a 2mL screw tube.
Add870µL Lysis master mix to 2mL screw tube. The final look:
Collect 20-50mg of sample to 2mL screw tube
Sample lysis
Put the 2mL screw tube on vortex for sample lysis, at 3200 rpm 0h 4m 0s
Centrifugation
Put 2mL screw tube in centrifuge for centrifugation, at this condition:10x g,25°C
DNA extraction
Add 350µL of isopropanol to the 1st well of 96 well plate
Add 50µL of magnetic beads (10mg/ml) to the 1st well of 96 deep well plate
Add 400µL of isopropanol to the 2nd well of 96 deep well plate
Add 300µL of 80% ethanol to the 3rd well of 96 deep well plate
Add 300µL of 95% ethanol to the 4th well of 96 deep well plate
Add 300µL of DDW to the 5th well of 96 deep well plate
Add 100µL of DEPC-treated water to the 6th well of 96 deep well plate
Add 300-500µL of the sample (lysate) from the 1.5mL centrifuged tube to the 1st well of 96 deep well plate
Put the prepared 96-deep well plate in the automated DNA extraction machine (ZiXpress 32) and set up the settings as below.
Program settings
The automated extraction program settings for ZiXpress 32.
| A | B | C | D | E | F | G |
|---|---|---|---|---|---|---|
| 1 | 0 | 10 | 1000 | 3 | 120 | 0 |
| 2 | 0 | 2 | 400 | 3 | 60 | 0 |
| 3 | 0 | 2 | 300 | 3 | 60 | 0 |
| 4 | 15 | 0 | 0 | 0 | 0 | 0 |
| 4 | 0 | 2 | 300 | 3 | 60 | 0 |
| 5 | 0 | 1 | 300 | 3 | 0 | 0 |
| 6 | 0 | 5 | 100 | 3 | 120 | 55 |
gDNA collection
After the extraction is done, put on the 96 magnetic plate to pellet the magnetic bead residues.
Collect 100µL of the eluted sample (avoid getting magnetic bead) as the DNA template for downstream experiments
