Jul 15, 2024

Public workspaceHigh Molecular Weight (HMW) DNA Extraction Protocol for Tissue Sample

DOI
dx.doi.org/10.17504/protocols.io.kqdg3xdrpg25/v1
  • Aswini Leela Loganathan1
  • 1Monash University Malaysia
Aswini Leela Loganathan
Open access
DOI: dx.doi.org/10.17504/protocols.io.kqdg3xdrpg25/v1
Protocol Citation: Aswini Leela Loganathan 2024. High Molecular Weight (HMW) DNA Extraction Protocol for Tissue Sample. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3xdrpg25/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: Working
We use this protocol and it's working
Created: January 22, 2024
Last Modified: July 15, 2024
Protocol Integer ID: 93868
Abstract
This is an organic extraction protocol used for high molecular DNA extraction for tissue samples. This protocol is suitable for obtaining gDNA for PacBio Sequel IIe library preparation and sequencing.
HMW DNA Extraction Method
HMW DNA Extraction Method
9h 12m 10s
Lysis the tissue sample.
Add Amount500 µL of lysis buffer to 50 - 150 mg of Sample of sample. Make sure the tissue sample has been finely cut.

Denatures and digest proteins that are subsequently hydrolyzed with Proteinase K.
Add Amount10 µL of Proteinase K and vortex for Duration00:00:10 .

10s
Incubate on a shaking incubator at Temperature55 °C for Duration02:00:00 ( or till dissolved).

2h
Remove RNA with RNAse .
Add Amount5 µL of RNAse and vortex briefly.

Incubate in a shaking incubator for at Temperature37 °C .

Partitioning of lipids and debris into an organic phase using P:C:IA.
Add Amount500 µL of P:C:IA (25:24:1). Vortex until an emulsion is formed.

Centrifuge at TemperatureRoom temperature for Duration00:10:00 at Centrifigation10000 x g .

10m
Pipette Amount200 µL of the aqueous layer into a new tube.

Neutralize the charges on the sugar-phosphate backbone of the DNA with Sodium Acetate.
Add 1/10th : Amount20 µL 3M Sodium Acetate. Vortex gently (avoid creating bubbles).

Precipitation Step.
Add 2 Vol : Amount440 µL of ice-cold 100% Ethanol. Mix gently and slowly by inverting the tube.

Incubate at Temperature-20 °C for Duration02:00:00 .

2h
Centrifuge at Centrifigation10000 x g, Room temperature, 00:02:00 .

2m
Wash Step.
Wash with ice-cold 70% ethanol.
Centrifuge for Duration00:02:00 at Centrifigation10000 x g .

2m
Repeat Wash Step (Step 13 and 14) 2 more times.
Air dry for at least Duration00:30:00 .

30m
Final elution.
Add Amount35 µL of elution buffer (EB) and mix gently using a wide bore tips or by tapping.

Storage.
Store the gDNA at Temperature4 °C