DNA extraction (BOMB)

Yin-Tse Huang, Tsu-Chun Hung

Published: 2023-04-19 DOI: 10.17504/protocols.io.n2bvj6mdnlk5/v4

Abstract

Add 200µL of 1mm beads to 1.5mL eppendorf tube

Add200µL of 0.5mm beads to 1.5mL eppendorf tube

Add870µL Lysis master mix to 1.5mL eppendorf tube

Note

In 4°C fridgeLysis master mix: 225 µL of TE buffer + 375 µL of lysis buffer + 270 µL of 10M ammonium acetate

Collect 10-20mg of sample to 1.5mL eppendorf tube

Put 1.5mL eppendorf tube in mixmill for sample crush, at this condition: 30 rpm/s, for 4mins 0h 4m 0s

Put 1.5mL eppendorf tube in centrifuge for centrifugation, at this condition:17.0x g,25°C

Add 350µL of isopropanol to the 1st well of 96 well plate

7.1.

Add 100µL of magnetic beads (10 mg/ml) to the 1st well of 96 deep well plate

Note

Vortex the bottle and pipetting before using magnetic beads; re-do vortex after adding to 3 samples to prevent set down of magnetic beads

7.2.

Add 200-300µL of the sample (lysate) from the 1.5mL centrifuged tube to the 1st well of 96 deep well plate

Note

Pipetting as much lysate as you can , as long as it's free of any cell debris (no solids in your tip) ADD at LAST

Add 400µL of isopropanol to the 2nd well of 96 deep well plate

Add 300µL of 80% enthanol to the 3rd well of 96 deep well plate

10.

Add 300µL of 80% enthanol to the 4th well of 96 deep well plate

11.

Add 300µL of DDW to the 5th well of 96 deep well plate

12.

Add 100µL of DEPC-treated water to the 6th well of 96 deep well plate

13.

Put the prepared 96 deep well plate in the automated DNA extraction machine and select the BOMB protocol

14.

After the extraction is done, collect 50-100µL of the eluted sample as the DNA template for downstream experiments