DNA extraction (BOMB)

Yin-Tse Huang, Tsu-Chun Hung

Published: 2022-07-04 DOI: 10.17504/protocols.io.261gen7ryg47/v1

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Abstract

Steps

Sample Collection

Add 200µL of 1mm beads to 1.5ml enppendorf tube

Add200µL of 0.5mm beads to 1.5ml enppendorf tube

Add 225µL of TE buffer to 2ml enppendorf tube

Note

TE buffer is in 4°C fridge

Add 375µL of lysis buffer to 2ml enppendorf tube

Note

Lysis buffer is in 4°C fridge

Add 267µL of 10M ammonium acetate to 2ml enppendorf tube

Note

10M ammonium acetate is in 4°C fridge

Collect 10-20mg of sample to 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

DNA purification

Add 350µL of isopropanol to the 1st well of 96 well plate

9.1.

Add 125µL 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

9.2.

Add 200µL of the sample (lysate) from the 2ml centrifuged tube to the 1st well of 96 deep well plate

Note

USUALLY ADD at LAST

10.

Add 400µL of isopropanol to the 2nd well of 96 deep well plate

11.

Add 300µL of 80% enthanol to the 3rd well of 96 deep well plate

12.

Add 300µL of 80% enthanol to the 4th well of 96 deep well plate

13.

Add 100µL of DD water to the 5th well of 96 deep well plate

14.

Put the prepared 96 deep well plate in the automated DNA extraction machine

15.

After the extraction is done, collect 100µL of the eluted sample as the DNA template for downstream experiments

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DNA extraction (BOMB)

Abstract

Steps

Sample Collection

Sample crush

Centrifugation

DNA purification

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