Jul 18, 2022
DNA extraction (BOMB_Soil)
Forked from DNA extraction (BOMB)
- 1KMU;
- 2Kaohsiung Medical College
Protocol Citation: Tsu-Chun Hung, Yin-Tse Huang, Hsin-Mao Wu 2022. DNA extraction (BOMB_Soil). protocols.io https://dx.doi.org/10.17504/protocols.io.q26g74e8kgwz/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: In development
We are still developing and optimizing this protocol
Created: July 18, 2022
Last Modified: September 13, 2023
Protocol Integer ID: 66907
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Abstract
DNA extraction (BOMB_Soil)
Materials
- P1000 pipette
- 96-deep well
- MagBeads
- TE buffer
- Lysis buffer
- Isopropanal
- 80 % Ethanol
- DEPC treated water
- ZiXpress 32 System
- 0.5mm zirconia beads
- 1.0mm zirconia beads
- 0.133M Ammonium acetate
- 0.06M Aluminium sulfate
Sample Collection
Sample Collection
3m
3m
Measuring 250 mg for a soil sample
Add 200 µL of 1mm beads to 2ml enppendorf tube
30s
Add200 µL of 0.5mm beads to 2ml enppendorf tube
30s
Add 225 µL of TE buffer to 2ml enppendorf tube
Note
TE buffer is in 4°C fridge
30s
Add 375 µL of lysis buffer to 2ml enppendorf tube
Note
Lysis buffer is in 4°C fridge
30s
Take 2ml enppendorf tube out of the laminar flow and transfer soil samples to the 2ml enppendorf tube
1m
Sample crush
Sample crush
4m
4m
Put 2ml eppendorf tube in mixmill for sample crush, at this condition: 30 rpm/s, for 4mins 00:04:00
4m
Centrifugation
Centrifugation
3m
3m
Put 2ml eppendorf tube in centrifuge for centrifugation, at this condition:17.0 x g, 25°C, 00:03:00
3m
Remove proteins and humic acid
Remove proteins and humic acid
Add 250 µL 0.133M Ammonium acetate in a new 2ml enppendorf tube
Transfer supernatant 300 µL of step 8 into 2ml enppendorf tube in step 9
Incubate on ice for 00:10:00
10m
Put 2ml eppendorf tube in centrifuge for centrifugation, at this condition:17.0 x g, 25°C, 00:03:00
3m
Add 200 µL 0.06M Aluminium sulfate in another 2ml enppendorf tube
Transfer step 12 supernatant 500 µL in step 13’s 2ml enppendorf tube
Incubate on ice for 00:10:00
10m
Put 2ml eppendorf tube in centrifuge for centrifugation, at this condition:17.0 x g, 25°C, 00:10:00
10m
DNA purification
DNA purification
37m 30s
37m 30s
Add 350 µL of isopropanol to the 1st well of 96 well plate
30s
Add 125 µL of magnetic beads (10 mg/ml) to the 1st well of 96 deep well plate
Note
Shake the bottle and pipetting before using magnetic beads
30s
Add 200 µL of the sample (lysate) from the 2ml centrifuged tube to the 1st well of 96 deep well plate
Note
USUALLY ADD at LAST
30s
Add 400 µL of isopropanol to the 2nd well of 96 deep well plate
30s
Add 300 µL of 80% enthanol to the 3rd well of 96 deep well plate
30s
Add 300 µL of 80% enthanol to the 4th well of 96 deep well plate
30s
Add 100 µL of DD water to the 5th well of 96 deep well plate
30s
Put the prepared 96 deep well plate in the automated DNA extraction machine
34m
After the extraction is done, collect 100 µL of the eluted sample as the DNA template for downstream experiments