Nuclei Isolation and High Molecular Weight DNA Extraction

Overview

Teaching: 20 minutes
Exercises: 2 days

Questions

• How to extract DNA from a tissue sample?

Objectives

• Extract DNA from a fresh or frozen tissue sample.

This nuclei isolation protocol is an adaptation of the one reported by Zhang et al. (1995). The HMW DNA extraction protocol is one recommended by 10X Genomics.

Preparation

First make and autoclave these component solutions

• 1M Tris Base
• 3M KCl
• 0.5M EDTA (pH 8.0)
• DI Water
• 5M NaCl

10x HB (Homogenization buffer)

Wash Buffer (AKA 1X HB)

• 1xHB, MUST be made fresh on Day 1

Lysis Buffer

• Use 3 mL per tube (step 12)

Proteinase K Solution

• MUST be made on Day 1 for step 13

Day 1:

1. Add ice-cold 1x HB buffer (~10mL:1g tissue) to 5-7 g of fresh (or frozen) tissue. Homogenize thoroughly using a blender. Conversely frozen tissue can be ground using a cold mortar and pestle and then mixed into the 1xHB buffer.

2. Transfer the blended/ground tissue to an ice-cold 500 mL beaker. Gently swirl the contents with a magnetic stir bar for 5 minutes. (We keep the beaker in an ice bath while swirling).

3. Add Triton X-100 to make the final concentration 0.5% (which would be 0.025 ul/1mL of 1xHB buffer). Swirl the contents for another 5 minutes.

4. Filter the contents into a new beaker, through 2 layers of cheesecloth and a layer of Miracloth, by squeezing gently with gloved hands.

5. Aliquot the filtrate into 14 mL round-bottom test tubes, filling them about 2/3 full. (use as many tubes as necessary – and make it an even number of tubes for centrifuging).

6. Pellet the homogenate by centrifugation in a swinging bucket rotor at 1800 g, at 4°C, for 20 minutes.

7. Carefully discard the supernatant. Add approximately 5 mL of ice-cold wash buffer to each tube. Gently re-suspend the pellet using a small soft-hair paintbrush soaked in wash buffer. Top each tube up with another 5 mL of ice-cold wash buffer (10 mL in total).

8. Centrifuge the tubes at 57 g, at 4°C, for 2 minutes. This step should pellet and remaining intact cells and tissue residue. Carefully transfer the supernatant to new 14 mL round-bottom tubes.

9. Centrifuge the tubes at 1,800 g, at 4°C, for 15 minutes in a swinging bucket rotor. The resulting pellet should be white and of a uniform consistency. Repeat steps 7 - 9 if the pellet is still discoloured green or looks to still have other tissue residue.

10. Discard the supernatant, gently re-suspend the pellet in 8-10 mL of wash buffer (using a paintbrush), and centrifuge at 1,800 g, at 4°C, for 15 minutes in a swinging bucket rotor. Repeat step 10 2-3 times, or until the pellet is white and washes have no effect of pellet quality. During these 2-3 wash steps, the pellets can be resuspended in 4-5 mL of wash buffer, and the contents of tubes can be combined, to eventually reduce the number of tubes to 2.

11. Following final wash, discard supernatant and remove all traces of supernatant using a pipette.

12. Add 3 mL of Lysis Buffer to each tube. Gently re-suspend the pellet by inversion ~20 times.

13. Add 0.2 mL of 10% SDS to each tube. Add 0.5 mL of Proteinase K solution to each tube. Gently mix by inversion 5-10 times.

14. Digest the cell lysate overnight (12-18 hours) at 37°C in a shaking incubator set at ~50 rpm. (keep tubes in a rack, with only a slight tilt upwards from horizontal).

Day 2:

1. Add 1.2 mL of 5M NaCl to each tube. Mix by gently inverting the tube ~5 times.

2. Centrifuge at ~1000g, at 4°C, for 15 minutes.

3. Use a wide-bore pipette tip to slowly transfer the supernatant to a new 14 mL round-bottom tube. Add 8 mL of absolute ethanol to each tube.

4. Gently rock the tubes and look for the presence of DNA (a gooey clear or slightly whitish precipitant). Using a wide-bore tip (or possibly a glass loop if there is enough DNA), transfer the DNA to a microcentrifuge tube.

5. Air-dry the pellets (time may vary depending on consistency and volume). Do not over-dry.

6. Resupend the pellet in 100 uL of sterile water. Allow the DNA to completely resuspend overnight before attempting quality/quantity checks. Add more water (and/or very gentle mixing) if required.

Key Points

• Tissue needs to be handled differently depends on storage temperature (fresh/frozen).

• Both 1xHB buffer and Proteinase K Solution need to be made fresh on the day of use.

• Only put even number of tubes into centrifuge.

• Do not overdry your pellets.