DNA extraction (BOMB_Soil)
Yin-Tse Huang, Tsu-Chun Hung, Hsin-Mao Wu
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Abstract
DNA extraction (BOMB_Soil)
Steps
Sample Collection
Measuring 250mg for a soil sample
Add 200µL of 1mm beads to 2ml enppendorf tube
Add200µL of 0.5mm beads to 2ml enppendorf tube
Add 225µL of TE buffer to 2ml enppendorf tube
Add 375µL of lysis buffer to 2ml enppendorf tube
Take 2ml enppendorf tube out of the laminar flow and transfer soil samples to the 2ml enppendorf tube
Sample crush
Put 2ml eppendorf tube in mixmill for sample crush, at this condition: 30 rpm/s, for 4mins 0h 4m 0s
Centrifugation
Put 2ml eppendorf tube in centrifuge for centrifugation, at this condition:17.0x g,25°C
Remove proteins and humic acid
Add 250µL 0.133M Ammonium acetate in a new 2ml enppendorf tube
Transfer supernatant 300µL of step 8 into 2ml enppendorf tube in step 9
Incubate on ice for 0h 10m 0s
Put 2ml eppendorf tube in centrifuge for centrifugation, at this condition:17.0x g,25°C
Add 200µL 0.06M Aluminium sulfate in another 2ml enppendorf tube
Transfer step 12 supernatant 500µL in step 13’s 2ml enppendorf tube
Incubate on ice for 0h 10m 0s
Put 2ml eppendorf tube in centrifuge for centrifugation, at this condition:17.0x g,25°C
DNA purification
Add 350µL of isopropanol to the 1st well of 96 well plate
Add 125µL of magnetic beads (10 mg/ml) to the 1st well of 96 deep well plate
Add 200µL of the sample (lysate) from the 2ml centrifuged tube to the 1st well of 96 deep well plate
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
Add 300µL of 80% enthanol to the 4th well of 96 deep well plate
Add 100µL of DD water to the 5th well of 96 deep well plate
Put the prepared 96 deep well plate in the automated DNA extraction machine
After the extraction is done, collect 100µL of the eluted sample as the DNA template for downstream experiments
