High molecular weight plant DNA extraction for PacBio HiFi sequencing

Prepare 400 ml NI buffer in a glass beaker and leave on ice.

On ice

Prepare 100 ml sorbitol buffer in two 50 ml Falcon tubes.

On ice

Affix nylon mesh on top of one empty 500 ml glass beaker with string or tape (or place Corning cell strainer on 50 ml Falcon tube).

Arrange liquid nitrogen and mortar and pestles on fume bench.

Grind fresh leaf tissue in liquid nitrogen and mortar and pestle 3 times to a fine powder and add ground tissue sample to NI buffer prepared at step 1. Grind 1-2 grams leaf tissue at a time and in total approx. 30 grams in this protocol.

On ice

Filter sample-NI buffer mix through 100 µm pore size nylon mesh/beaker prepared at step 3. Keep all solutions on ice during filtering.

On ice

Divide filtrate equally to ten 50 ml Falcon tubes kept on ice.

On ice

Add 1/20^th volume of 10% Triton X-100/NIB to tubes prepared at step 7. Gently mix by inverting tubes.

On ice

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

2000x g,4°C

Discard supernatant gently by decantation, without disturbing or losing pellet.

Add 10 ml sorbitol buffer to each tube and mix gently.

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

3000x g,4°C

Discard supernatant by decantation (Optionally, repeat sorbitol buffer wash until supernatant is clear).

To remove sorbitol buffer completely, invert tubes on dry tissue briefly but take care not to lose pellet. Pellet of nuclei and small cells remaining in tubes can now be frozen in liquid nitrogen and stored at -80 °C.

-80°C

[SAFE STOP POINT for at least a few days]

Add 3 ml CTAB lysis buffer directly to frozen pellet, a pinch of PVPP, and 12 µl RNase A to each tube and mix well by gently pipetting with wide-bore tips. Incubate at 58 °C for 20 minutes.

58°C

Add 60 µl proteinase K to each tube. Incubate for more than 3 hours, but less than 5 hours, at 58 °C. Occasionally shake tubes gently.

58°C

Centrifuge at 4,400 × g for 10 minutes at room temperature. Collect clear lysate to new 50 ml Falcon tube avoiding any cell debris.

4400x g

To maximize lysate recovery, move remained debris/lysate to 2 ml tubes and centrifuge at 11,000 rpm for 5 minutes. Move clear lysate to same tube at step 17. In total approx. 30 ml lysate can be obtained.

11000rpm

Adjust lysate with 0.25 N HCl to between pH 7.0 - 7.5. Check pH with pH indicator strips. Add 1 ml or less 0.25N HCl at a time and check with pH paper each time.

Divide lysate to two 50 ml Falcon tubes. Add equal volume of Milli-Q water.

NOTE: Do not centrifuge tubes once water is added. Low salt condition tends to promote formation of a CTAB-DNA solidified complex.

Proceed with Qiagen Genomic-tip 100/G following the manufacturer`s protocol. Set up six empty 50 ml Falcon tubes, labelled “QBT”, “Sample”, “QC1”, “QC2”, “QC3”, “Final DNA”. Set up three sets of each.

Set water bath to 50 °C and prewarm buffer QF.

50°C

Place Genomic-tip 100/G column on 50 ml Falcon tube labelled “QBT”. Equilibrate Genomic-tip 100/G column with 4 ml buffer QBT. Allow buffer to flow through column completely by gravity.

Move Genomic-tip 100/G to next Falcon tube labelled “Sample”. Load one third of lysate (approx. 20 ml) obtained at step 20 to one Genomic-tip 100/G. Allow lysate to flow through column completely by gravity.

Move Genomic-tip 100/G to tube labelled “QC1”. Load 7.5 ml buffer QC onto column. Allow buffer to flow through column completely by gravity.

Repeat buffer QC step two more times, on tubes labelled “QC2” and “QC3”. In total, DNA in Genomic-tip 100/G column should be washed three times with buffer QC.

For final DNA elution, apply 5 ml QF buffer prewarmed to 50 °C to each Genomic-tip 100/G column.

50°C

Divide eluted DNA in 1 ml aliquots to 2 ml tubes. Add 0.7 volume (0.7 ml) ice-cold isopropanol. Gently invert and mix and leave tubes at -20 °C overnight.

-20°C

[SAFE STOP POINT for at least a few days]

Centrifuge tubes at 11,000 rpm for 10 minutes.

11000rpm

Discard supernatant and add 1 ml 70% ethanol.

Centrifuge at 11,000 rpm for 10 minutes.

11000rpm

Discard supernatant and air-dry pellet by inverting tubes on clean tissue. Warming tubes at 37 °C for 10 minutes speeds up evaporation of ethanol, but do not over-dry.

Add 15-20 µl Low (0.1 ×) TE buffer.

Incubate tubes at 50 °C, and 300 rpm for 1 hour. Collect eluted DNA to 1.5 ml LoBind Eppendorf tube.

50°C

Add 15-20 µl Low (0.1 ×) TE buffer for 2^ndelution.

Incubate tubes at 50 °C, and 300 rpm for 1 hour. Collect DNA elute to 1.5 ml LoBind Eppendorf tube. Keeping 1^stand 2^nd DNA elution in separate tubes is recommended.

50°C

Proceed to DNA quality control. DNA quantification with Qubit and Nanodrop. For DNA quality, obtain A260/A280 and A260/A230 values with Nanodrop. Examine DNA fragment size distribution with TapeStation or FemtoPulse.