Sep 22, 2023
DNA extraction v9.0 (modified BOMB)
Forked from DNA extraction (BOMB)
- 1Kaohsiung Medical University;
- 2KMU
Protocol Citation: Guan Jie Phang, Tsu-Chun Hung, Yin-Tse Huang 2023. DNA extraction v9.0 (modified BOMB). protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwdbb7lmk/v1
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 14, 2023
Last Modified: September 22, 2023
Protocol Integer ID: 87762
Abstract
DNA extraction using yttria-stabilized zirconia beads lysing and automated magnetic bead-based extraction.
Materials
1. Lysis master mix 870 ml (870 μl/sample)
| Chemical | Volume | |
| TE buffer | 225 μl | |
| Lysis buffer | 375 μl | |
| 10M Ammonium acetate | 270 μl |
2. TE buffer stocks 225 ml
| Chemical | Volume | Notes | |
| 1M Tris-HCl pH8.0 | 2.25 ml | 10mM | |
| 1M EDTA | 0.225 ml | 1mM | |
| ddH2O | adjust the volume with water to 225 ml | ||
Note
Prepare 1M Tris-HCl and 1M EDTA first.
| Chemical | Mass | Total volume | |
| Tris-HCl | 7.88 g | 50 ml | |
| EDTA | 14.61 | 50 ml |
Note
adjust the pH of EDTA with solid NaOH or 1M NaOH to 8.0. The undissolved EDTA will be dissolved entirely when the pH reaches 8.0.
3. Lysis buffer stocks 375 ml
| Chemical | Volume/Mass | Notes | |
| Guanidine thiocyanate (GITC) | 177.3 g | 4M | |
| 1M Tris HCl pH8.0 | 18.75 ml | 50mM | |
| Sodium Lauryl Sulfate | 3.75 g | 0.5 g | |
| 1M EDTA | 7.5 ml | 20mM | |
| ddH2O | adjust the pH with HCl to 7.6–8.0 and the volume with water to 375 ml | ||
4. 10M Ammonium acetate 270 ml
| Chemical | Volume/Mass | |
| Ammonium acetate | 208.116 g | |
| ddH2O | adjust the volume with water to 270 ml |
Note
Ammonium acetate is hygroscopic. Do not add more than half of the water needed to dissolve it.
Sample Collection
Sample Collection
3m
Add 200 µL of 0.5 mm beads to a 2mL screw tube.
30s
Add 200 µL of 1 mm beads to a 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 lysis
Sample lysis
4m
Put the 2mL screw tube on vortex for sample lysis, at 3200 rpm 00:04:00
Note
Remember to balance if you have odd number of samples
4m
Centrifugation
Centrifugation
3m
Put 2mL screw tube in centrifuge for centrifugation, at this condition:10 x g, 25°C, 00:03:00
3m
DNA extraction
DNA extraction
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 (ZiXpress 32) and set up the settings as below.
34m
Program settings
Program settings
37m 30s
The automated extraction program settings for ZiXpress 32.
| Well no. | Standby(mins) | Mix(mins) | Volume(μl) | Mix speed | Mag(s) | Temp.(ºC) | |
| 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
gDNA collection
37m 30s
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