High Molecular Weight DNA extraction for long-read sequencing v.1

Prepare 200 ml Nuclei isolation buffer (NIB) and leave on ice.

On ice

Prepare 20 ml NIB base-Triton mix and leave on ice.

On ice

Prepare 60 ml Sorbitol buffer.

Prepare liquid nitrogen in a suitable dewar. Place mortar and pestle in fume hood.

Prepare six 50 ml Falcon tubes on ice and place 100 μm gauge cell strainers (Nylon Meshes) on top.

On ice

Prepare the other set of six 50 ml Falcon tube layered with 40 μm gauge cell strainers, leave on ice.

On ice

Pre-cool the pestle and mortar with liquid nitrogen.

Cut 1-2g of fresh leaf tissue from living plants and place in liquid nitrogen to shock-freeze.

Add frozen tissue to pre-cooled mortar held in a Styrofoam mortar holder.

Add some liquid nitrogen and grind carefully with pestle as the liquid nitrogen evaporates.

Once the liquid nitrogen has evaporated, grind more vigorously for 30 seconds, until the material is a fine powder.

Do not allow the tissue powder to thaw.

Add liquid nitrogen (enough to cover the tissue) and repeat step10 two more times.

Transfer the ground tissue to a beaker of cold NIB placed on ice with a pre-cooled spatula and mix by gently swirling (Fig. 2). Keep on ice.

On ice

Repeat the above steps 7 to 12 until all the leaf tissue is ground. The suspension should be smooth with no clumps of leaf material.

Filtering to remove particulate material. Proceed steps on ice.

On ice

Filter the NIB/sample suspension through 100 μm nylon mesh filter by gravity (see step 5). Do not force the sample through the filter. Replace filters when it is clogged.

On ice

Filter the 100 μm mesh filtrate through the 40 μm nylon mesh (see step 6). Replace filters when it is clogged.

On ice

Divide the filtrate of step 14.2 equally into six Falcon tubes, with 40– 50 ml in each.

On ice

Add 1/20th volume of NIB base-Triton mix, close the tubes with lids and mix the solution by gently swirling and inverting the tubes.

Centrifuge at 2,000 × g for 10 minutes, if possible at 4 °C.

2000x g

Carefully discard supernatant without dislodging the pellet.

Add 10 ml Sorbitol buffer to the pellet in each tube and swirl the tubes to mix well.

Centrifuge at 3,000 × g for 10 minutes, if possible at 4 °C.

3000x g

Discard supernatant leaving the pellet undisturbed. Invert the tubes on tissue to remove traces of buffer without losing the pellet.

Set water bath to 50 °C.

50°C

Add 5 ml of QIAGEN Buffer G2 to each 50 ml Falcon tube containing the nuclei/ cells and gently resuspend the pellet.

Add 0.1% β-mercaptoethanol (5 μl) and a pinch of PVPP to the tube.

Swirl the tube to mix well.

Incubate the tubes at 50 °C for 5 minutes.

50°C

Add 10 μl RNase A or 50 μl RNase A/T1 Mix to the tube.

Gently swirl the tubes to mix.

Incubate the tubes at 50 °C for 5 minutes for RNase A, or 30 minutes for RNase A/T1 Mix.

50°C

Add 100 µl QIAGEN Proteinase K to the tube.

Swirl the tubes to mix well.

Incubate the tubes at 50 °C in the water bath, for > 5 hours or overnight.

50°C

Set up for QIAGEN Genomic-tip 100/G.

Set up Genomic-tip kit: Label 5 × 50 ml Falcon tubes without lids as follows: 1. QBT, 2. Sample, 3. QC 1, 4. QC 2,

5. Final sample.

Pre-heat Buffer QF to 50 °C in the water bath.

50°C

To obtain the clear lysate, centrifuge the 50 ml Falcon tubes containing nuclei and cell lysate from step 28 at 3,000 × g for 10 minutes.

3000x g

Equilibrating a Genomic-tip 100/G column.

Place the column mounted in the column holder on top of the tube labelled ' 1.  QBT' (see Fig.4).

Add 4 ml Buffer QBT to the column.

Allow the column to empty by gravity flow, do not force the remaining buffer through.

Move the column to the ' 2. Sample’ tube and apply 5 ml of the clear lysate (supernatant obtained at step 30) to the equilibrated Genomic-tip.

Allow the column to empty by gravity flow.

Wash Genomic-tip 100/G columns with Buffer QC .

Move the column to the ‘ 3. QC 1’ tube and add 7.5 ml of Buffer QC.

Allow Buffer QC to move through the column by gravity flow.

Move the column to the ‘ 4. QC 2’ tube and add 7.5 ml Buffer QC.

Allow Buffer QC to move through the column by gravity flow.

Eluting the genomic DNA from the Genomic-tip 100/G column.

Move the column to the ‘ 5. Final Sample’ tube and add 5 ml of Buffer QF pre-warmed to 50 °C.

Allow Buffer QF to flow through the column by gravity.

Store the eluted DNA sample on ice.

Transfer the DNA sample obtained at step 34 to a 2 ml Eppendorf tube.

Add 1 ml sample per tube (totaling 30 tubes from 30 ml elution buffer [5 ml Buffer QF × 6 tubes] at the step 34 in our example).

Add 700 μl of ice-cold isopropanol to each tube and gently invert the tubes to mix.

Leave samples at −20 °C for > 2 hours or overnight.

-20°C

Centrifuge tubes at 11,000 rpm for 10 minutes.

11000rpm

Discard the supernatant.

Add 400 µl ice cold 70% ethanol to the tubes.

Centrifuge the tubes at 11,000 rpm for 10 minutes.

11000rpm

Discard the supernatant by tipping the tube, with the pellet on the upper side, over a clean glass beaker or a Falcon tube. 

Invert tubes on tissue to remove residual ethanol.

Air dry pellet for approximately 10 – 20 minutes to remove all trace of ethanol.

Add 50 μl TE buffer (or any desired buffer for the downstream application) to the each tube.

Leave at 50 °C for 1 hour.

50°C

Collect the DNA in two 2ml tubes; i.e., from the 30 tubes with 50 μl DNA sample in each (see step 44), 1.5 ml of DNA sample is obtained, and thus add 750 μl of DNA sample to each tube.

2nd Precipitation to concentrate DNA:

Add 1/10th volume (75 μl) of 3 M NaOAc and an equal volume (825 μl)of ice-cold isopropanol to each tube.

Leave the tubes at −20 °C, for more than 1 hour to overnight.

-20°C

Centrifuge the tubes at 11,000 rpm for 10 minutes.

11000rpm

Discard the supernatant and Invert tubes on tissue to remove residual ethanol.

Air-dry pellet for 10 – 20 minutes. Do not overdry the DNA samples.

Elute the DNA pellet in an appropriate volume of elution buffer depending on the downstream application.

Leave the tubes at 50 °C, 300 rpm in a ThermoMixer for 1 hour.

50°C

Assessing DNA integrity: Perform quality checks for DNA integrity by 0.8% agarose gel electrophoresis or Agilent Technologies TapeStation Genomic DNA ScreenTape.

Assessing DNA quality: Perform a DNA quality assessment for OD values with a spectrophotometer such as NanoDrop or DeNovix.

Assessing DNA quantity: For NGS applications, DNA quantity must be assessed using a fluorometer such as the Qubit or DeNovix.