Sep 10, 2023

DNA extraction (BOMB) V.8

DOI
https://dx.doi.org/10.17504/protocols.io.n2bvj6mdnlk5/v8
  • 1KMU
Yin-Tse Huang
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DOI: https://dx.doi.org/10.17504/protocols.io.n2bvj6mdnlk5/v8
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
Keywords: dna extraction, bomb, dna, extraction
Abstract
DNA extraction (BOMB)
Materials
1. Lysis master mix (870 uL/sample)

AB
TE buffer225 uL
Lysis buffer375 uL
Ammonium acetate270 uL
2. TE buffer

AB
Tris HCl pH8.010mM
EDTA1mM
3. Lysis buffer

AB
GITC4M
Tris HCl pH8.050mM
SDS0.5g
EDTA20mM


Sample Collection
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
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
3m
Put 2mL screw tube in centrifuge for centrifugation, at this condition:10 x g, 25°C, 00:03:00
3m
DNA purification
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