DNA extraction (BOMB)

Yin-Tse Huang, Tsu-Chun Hung

Published: 2022-07-02 DOI: 10.17504/protocols.io.x54v9ywq1g3e/v1
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Abstract

DNA extraction (BOMB)

Steps

Sample Collection

1.

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

2.

Add200µL of 0.5mm beads to 1.5ml enppendorf tube

3.

Add 225µL of TE buffer to 1.5ml enppendorf tube

Note
TE buffer is in 4°C fridge

4.

Add 375µL of lysis buffer to 1.5ml enppendorf tube

Note
Lysis buffer is in 4°C fridge

5.

Add 267µL of 10M ammonium acetate to 1.5ml enppendorf tube

Note
10M ammonium acetate is in 4°C fridge

6.

Collect 10-20mg of sample to 1.5ml enppendorf tube

Sample crush

7.

Put 1.5ml eppendorf tube in mixmill for sample crush, at this condition: 30 rpm/s, for 4mins 0h 4m 0s

Centrifugation

8.

Put 1.5ml eppendorf tube in centrifuge for centrifugation, at this condition:17.0x g,25°C

DNA purification

9.

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 sample (lysate) from 15ml centrifuge microtube 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 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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