# Extraction of high-molecular weight (HMW) DNA

[EcoEvoDevoLab](https://hackmd.io/@EcoEvoDevoLab/AngeliniLab)

Updated 8 June 2023


## Protocol

This protocol is intended to obtain DNA suitable for long-read sequencing. It is based on a [protocol](https://www.protocols.io/view/hmw-dna-extraction-for-insects-rm7vzpj2lx1w/v1) recommend by Oxford Nanopore.

Because DNA is a very long polymer, isolating very large fragements can be difficult. Care should be taken not be vortex samples or to pass the DNA quickly through pipette tips, all of which can shear long DNAs. Live tissue is also the best source.

### Materials

- liquid nitrogen
- dry ice
- TisueLyser beads (e.g. [QIAGEN catalog number 69997](https://www.qiagen.com/us/products/beads/))
- Genomic-tip 20/G ([QIAGEN catalog number 10223](https://www.qiagen.com/us/products/qiagen-genomic-tips/))
- RNase A (e.g. [QIAGEN catalog number 19101](https://www.qiagen.com/us/products/rnase-a/))
- Proteinase K (e.g. [QIAGEN catalog number 19131](https://www.qiagen.com/us/products/qiagen-protease-and-proteinase-k/))
- Isopropanol
- 70% Ethanol (nuclease-free, -20˚C)

### Procedure

#### Before starting

- **Weigh** and **photograph** insects before using them for extractions. For genomic projects be sure to determine their species, sex and geographic origin.
- **Chill the TissueLyser tube holder** on dry ice.
- **Prepare the lysis buffer** by adding 1.5 µL of RNase A (100 mg/mL) to 1438.5 µL of Buffer G2 per sample.

#### Homogenization

- **Place insects in a 1.7 mL micro centrifuge tube** with **two 3-mm disruptor beads** (RNase-free)
- **Flash freeze** in liquid nitrogen.
- **Attach the tube holder** to the TissueLyser .
- **Disrupt** the tissue for 90 seconds at 20 Hz.

:::warning
The goal of this step is to pulverise the insects without allowing them to thaw. Grinding time requires optimisation to balance DNA yield against physical damage to the DNA.
:::

- After disruption, immediately **return the tubes to liquid nitrogen**. Alternatively, **store at -80°C** until you're ready to continue.

#### Lysis

- Remove the powdered sample from liquid nitrogen (or the -80°C freezer) and immediately **add 1440 µL of lysis buffer**. (Be sure RNAase A has been added.)
- **Incubate at 37°C for 30 minutes** with inversion.
- **Add 60 µL of Proteinase K** (20 mg/mL) and incubate at **50°C for 2 hours** with inversion.

#### Initial seperation

- **Centrifuge** at max speed for 20 minutes to pellet the debris.
- Meanwhile, **add 1 mL Buffer QBT to a QIAGEN Genomic-tip 20/G**. (This will allow it to equilibrate. Wait for the Genomic-tip to drain by gravity.)
- After centrifugation, gently **transfer 1200 µL of supernatant** into a 15 mL Falcon tube.
- **Dilute the lysate to roughly 3 mL**. (The volume doesn't matter, but if the sample is more dilute it will run through the column faster.)

#### DNA binding

- Carefully **apply the lysate to the Genomic-tip**. Do not vortex the sample. Gently invert it a few times to mix, then pour as much as you can from the 15 mL tube into the Genomic-tip. Pulse spin the 15 mL tube to collect any remaining lysate, and slowly pipette it into the Genomic-tip with a P-1000. Wait for the Genomic-tip to drain by gravity. Draining can take a long time, particularly if the sample is viscous (30–60 minutes), but resist the urge to apply pressure.
- **Wash** the Genomic-tip with **1 mL Buffer QC**.
- **Repeat three times** for a total of 4 mL of Buffer QC.

#### DNA recovery

- **Elute** the DNA into a new 15 mL tube with 2 mL of Buffer QF. Wait patiently for the tip to drain.
- After elution **slowly pipette 667 µL of eluate into three new 1.7 mL tubes** with a P-1000.

:::info
Splitting the sample into three is necessary if the lab doesn't have a fast centrifuge for 15 mL tubes.
:::

- Add 467 µL of room-temperature **isopropanol** to each tube, mix by inversion about 10 times.
- Centrifuge for 20 min at 15,000×g at 4°C to pellet DNA.
- **Slowly pour off the supernatant**. (The pellet can be hard to see! Keep the supernatant from this step and the ethanol wash later until the final yield has been tested.) Pulse-spin the tubes, and remove any remaining traces of supernatant with a P-10.
- **Add 1 mL of 70% ethanol at -20˚C**. Invert to mix.
- **Centrifuge** at 15,000×g for 10 min at 4°C.
- **Pour off the ethanol**, pulse-spin the tubes, and remove any remaining traces of ethanol.
- **Repeat** the 70% ethanol wash.
- **Air dry the pellet** briefly. It can be difficult to resuspend, so don't leave it too long. Try to remove all the ethanol with a P-10, and then watch the tubes until the traces evaporate.
- **Resuspend** each pellet in **55 µL of Qiagen Buffer EB** (which is simply Tris HCl, pH 8). Leave the tubes **overnight at room temperature with inversion** to resuspend. Do not pipette the sample up and down!

#### Quality control

- Use the **NanoDrop spec** to measure the concentration of the DNA (ng/µl). Record this information in your notebook. Also note the A~260~/A~280~ and A~260~/A~230~ ratios for the sample. (Pure DNA should have an A~260~/A~280~ ratio > 1.8.)
- Run out 1 µL of sample on a **0.7% agarose gel** at 80 V for 60 minutes to estimate the molecular weight. Use a high-molecular weight DNA ladder for comparison.

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[EcoEvoDevo Lab](https://hackmd.io/@EcoEvoDevoLab/AngeliniLab)