DNA extraction (BOMB)

Yin-Tse Huang, Tsu-Chun Hung

Published: 2023-06-12 DOI: 10.17504/protocols.io.n2bvj6mdnlk5/v6

AI 解读

Abstract

Steps

DNA purification

0.20.

Add 100µL of magnetic beads (10 mg/ml) to the 1st well of 96 deep well plate

Note

Vortex the bottle and pipetting before using magnetic beads; re-do vortex after adding to 3 samples to prevent set down of magnetic beads

Sample Collection

Add 200µL of 0.5 mm beads to 2mL screw tube

Add200µL of 1 mm beads to 2mL screw tube

Add870µL Lysis master mix to 2mL screw tube. The final look:

Note

In 11F, 4°C fridgeLysis master mix: 225 µL of TE buffer + 375 µL of lysis buffer + 270 µL of 10M ammonium acetate

Collect 20-50mg 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.

Sample crush

Put the 2mL screw tube in mixmill for sample crush, at 3200 rpm 0h 4m 0s

Note

Remember to balance if you have odd number of samples

Centrifugation

Put 2mL screw tube in centrifuge for centrifugation, at this condition:10x g,25°C

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

Add 100µL of magnetic beads (10mg/ml) to the 1st well of 96 deep well plate

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

10.

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

11.

Add 300µL of 95% ethanol to the 4th well of 96 deep well plate

12.

Add 300µL of DDW to the 5th well of 96 deep well plate

13.

Add 100µL of DEPC-treated water to the 6th well of 96 deep well plate

14.

Add 300-500µL 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)

15.

Put the prepared 96 deep well plate in the automated DNA extraction machine and select the BOMB protocol

16.

After the extraction is done, put on the 96 magnetic plate to pellet the magnetic bead residues.

17.

Collect 100µL of the eluted sample (avoid getting magnetic bead) as the DNA template for downstream experiments

FLEP-seq: simultaneous detection of RNA polymerase II position, splicing status, polyadenylation site and poly(A) tail length at genome-wide scale by single-molecule nascent RNA sequencing

DNA extraction (BOMB)

Abstract

Steps

DNA purification

Sample Collection

Sample crush

Centrifugation

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