Sep 10, 2023
Version 8
DNA extraction (BOMB) V.8
- 1KMU
Protocol Citation: Yin-Tse Huang, Tsu-Chun Hung 2023. DNA extraction (BOMB). protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvj6mdnlk5/v8Version created by Yin-Tse Huang
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 09, 2023
Last Modified: September 13, 2023
Protocol Integer ID: 87598
Abstract
DNA extraction (BOMB)
Materials
1. Lysis master mix (870 uL/sample)
| A | B | |
| TE buffer | 225 uL | |
| Lysis buffer | 375 uL | |
| Ammonium acetate | 270 uL |
2. TE buffer
| A | B | |
| Tris HCl pH8.0 | 10mM | |
| EDTA | 1mM |
3. Lysis buffer
| A | B | |
| GITC | 4M | |
| Tris HCl pH8.0 | 50mM | |
| SDS | 0.5g | |
| EDTA | 20mM |
Sample Collection
Sample Collection
3m
3m
Add 200 µL of 0.5 mm beads to 2mL screw tube
30s
Add200 µL of 1 mm beads to 2mL screw tube
30s
Add870 µL Lysis master mix to 2mL screw tube. The final look:
Note
In 11F, 4°C fridge
Lysis master mix: 225 µL of TE buffer + 375 µL of lysis buffer + 270 µL of 10M ammonium acetate
30s
Collect 20-50 mg of sample to 2mL screw tube
Note
You can collect up to 100 mg of sample if you can until you bump into the low DNA quality or PCR success rate; by then it means too many inhibitors in the sample and you have to lower the input.
1m
Sample crush
Sample crush
4m
4m
Put the 2mL screw tube in mixmill for sample crush, at 3200 rpm 00:04:00
Note
Remember to balance if you have odd number of samples
4m
Centrifugation
Centrifugation
3m
3m
Put 2mL screw tube in centrifuge for centrifugation, at this condition:10 x g, 25°C, 00:03:00
3m
DNA purification
DNA purification
37m 30s
37m 30s
Add 350 µL of isopropanol to the 1st well of 96 well plate
30s
Add 50 µL of magnetic beads (10mg/ml) to the 1st well of 96 deep well plate
Add 400 µL of isopropanol to the 2nd well of 96 deep well plate
30s
Add 300 µL of 80% ethanol to the 3rd well of 96 deep well plate
30s
Add 300 µL of 95% ethanol to the 4th well of 96 deep well plate
30s
Add 300 µL of DDW to the 5th well of 96 deep well plate
Add 100 µL of DEPC-treated water to the 6th well of 96 deep well plate
30s
Add 300-500 µL of the sample (lysate) from the 1.5mL centrifuged tube to the 1st well of 96 deep well plate
Note
Pipetting as many lysate as you can, as long as it's free of any cell debris (no solids in your tip)
30s
Put the prepared 96 deep well plate in the automated DNA extraction machine and select the BOMB protocol
34m
After the extraction is done, put on the 96 magnetic plate to pellet the magnetic bead residues.
Collect 100 µL of the eluted sample (avoid getting magnetic bead) as the DNA template for downstream experiments