Isolating nuclei
from planttissue using LN2
disruption
Procedure & checklist© 2024 PacBio. All rights reserved. Research use only. Not for use in diagnostic procedures.
102-574-800 REV03 JUN2024

Page 2Equipment and reagent list
Equipment/reagent

Manufacturer (part number)

Liquid nitrogen-cooled mortar

Fisher Scientific (H372600000)

Porcelain pestle

Fisher Scientific (FB961N)

Refrigerated centrifuge

Eppendorf (022625101)

Micro-centrifuge

Eppendorf (5404000413)

Tube rotator, shaker, or platform rocker

Fisher Scientific Mini-Tube Rotator (05-450-127)

Steriflip (20 µm pore size, 50 mL process volume)

Millipore (SCNY00020)

Small nylon or synthetic paintbrush

Any major lab supplier (MLS)

1.5 mL Protein LoBind micro-centrifuge tubes

Eppendorf (022431081)

50 mL conical tubes

ThermoFisher (339652)

2-mercaptoethanol, 14 M

Sigma-Aldrich (M3148)

Triton X-100

Sigma-Aldrich (X100)

Trizma baseSigma-Aldrich (T4661)

Potassium chloride (KCl)Sigma-Aldrich (P9541)

Ethylenediaminetetraacetic acid (EDTA), 0.5 M, pH 8.0

ThermoFisher (15575020)

Spermidine trihydrochloride

Sigma-Aldrich (S2501)

Spermine tetrahydrochloride

Sigma-Aldrich (S1141)

SucroseSigma-Aldrich (S0389)

Sodium hydroxide (NaOH), 10 M

Sigma-Aldrich (72068)

Polyvinylpyrrolidone (MW ~360 kD) (PVP360)

Sigma-Aldrich (PVP360)

Ultra-pure water

Any MLS

Liquid nitrogen (LN2)

Any MLS

pH meter

Any MLS© 2024 PacBio. All rights reserved. Research use only. Not for use in diagnostic procedures.
102-574-800 REV03 JUN2024

Page 3For all protocols
Eppendorf Protein LoBind tubes (Eppendorf #022431081) are highly recommended for all extractions to reduce protein
contamination from tube carryover. Protein LoBind tubes are more effective in reducing carryover contamination thanDNA LoBind tubes or other tubes and will result in improved UV purity.

Safety precautions
Refer to the Safety Data Sheet (SDS) for information on reagent hazards and protocols for safe handling, use, storage,and disposal.

Buffer preparation
Prepare the following buffers prior to beginning nuclei isolation.

Buffer 10X HB – homogenization buffer stock (500 mL)
Add the following reagents to a clean beaker and stir until dissolved:
Reagent

AmountFinal concentration

Trizma6.06 g0.1 M

KCl

29.8 g

0.8 M

EDTA (0.5 M)

100 mL

0.1 M

Spermidine

1.28 g

17 mM

Spermine

1.74 g

17 mM

Bring to a final volume of 500 mL with ultra pure water and adjust pH to 9 with 10 M NaOH. Store at 4°C in a glass
bottle for up to 1 year.

Buffer 1X HB – homogenization buffer (1 L)
Add the following reagents to a clean beaker and stir until dissolved:
Reagent

AmountBuffer 10X HB

100 mL

Sucrose171.2 gBring to a final volume of 1 L with ultra-pure water. Store at 4°C in a glass bottle for 3 months.
© 2024 PacBio. All rights reserved. Research use only. Not for use in diagnostic procedures.
102-574-800 REV03 JUN2024

Page 4

Buffer TSB – triton-sucrose buffer (100 mL)
Add the following reagents to a clean beaker and stir until dissolved:
Reagent

Amount

Triton X-100

20 mL

Buffer 10X HB

10 mL

Sucrose17.1 gBring to a final volume of 100 mL with ultra-pure water. Store at 4°C in a glass bottle for up to 1 year.

Buffer NIB – nuclei isolation buffer (50 mL)
Note: Make 50 mL of Buffer NIB per gram of tissue being processed. Buffer NIB can be stored at 4°C for up to one
week prior to the addition of 2-mercaptoethanol.
Note: PVP360 dissolves slowly so please allow at least 2 h to prepare Buffer NIB before beginning the nuclei isolation
protocol.
Add the following reagents to a clean bottle and stir or use end-over-end mixing until dissolved:

Reagent

AmountBuffer 1X HB

48.75 mL

Buffer TSB

1.25 mL

PVP3600.5 gUse immediately or store at 4°C for up to one week.

© 2024 PacBio. All rights reserved. Research use only. Not for use in diagnostic procedures.
102-574-800 REV03 JUN2024

Page 5Nuclei isolation protocol
This protocol describes the isolation of nuclei from plant tissues for HMW DNA extraction using the Nanobind plant
nuclei kit (102-302-000). This protocol has been specifically optimized for HiFi sequencing. It is based on the protocol
described in Workman et al. protocol exchange (2018) (DOI:10.1038/protex.2018.059).

Input requirements
Amount: 1-5 g of plant tissue. Input will vary by plant type, age, and growing conditions.
• We recommend initially testing this protocol with 1 g of tissue. After evaluating the outcome from 1 g of tissue, theinput mass can be increased or decreased based on the needs of the user.
• The maximum tissue mass we recommend processing at once using liquid nitrogen (LN2) grinding is 5 g.
• The amount of plant tissue required will depend on species, age, and nuclei isolation efficiency. Therefore, the input
mass may need to be adjusted to achieve suitable DNA yield and purity.
• Young leaves, preferably grown in a greenhouse or growth chamber, will produce the highest quantity and quality ofDNA. Mature plants grown in more natural environments tend to require larger tissue inputs and more wash steps
to obtain equivalent DNA yields and purity.
• This protocol has been validated on a wide range of tissues, including leaf tissues from pepper, sugar beet,lavender, banana, arabidopsis, wheat, fescue, tomato, and spinach.
• This protocol is suitable for fresh or frozen plant tissues.

Processing tips
Tissue disruption with TissueRuptor is faster and typically results in higher extraction yields than LN2 grinding. For
some plant species, LN2 grinding may result in improved DNA size. It is recommended that users start with theTissueRuptor protocol (Procedure & checklist – Isolating nuclei from plant tissue using TissueRuptor disruption).

Nuclei isolation – LN2 plant tissue1.

Add 125 µL of 2-mercaptoethanol (14 M) to 50 mL of Buffer NIB and store on ice
throughout use.
• This protocol should be performed under a fume hood to avoid inhalation of 2mercaptoethanol.
• If >1 g of tissue is being processed, add 125 µL of 2-mercaptoethanol (14 M) per 50
mL Buffer NIB.

2.

Using a liquid nitrogen (LN2)-cooled mortar and pestle, grind 1-5 g of frozen planttissue into a flour-like powder. This requires >25 min of manual grinding.

© 2024 PacBio. All rights reserved. Research use only. Not for use in diagnostic procedures.
102-574-800 REV03 JUN2024

Quick tip
Grinding in liquid nitrogen forat least 25 min is critical formaximizing DNA recovery andpurity for most plant tissues. Ifin doubt, grind more!

Page 6

• Plant tissue must remain frozen during grinding. If an LN2-cooled mortar and pestle
is not available, LN2 must be continuously added throughout grinding to keep thetissue frozen.
• Tissue should be ground into an extremely fine powder with flour-like consistency.
• Insufficient grinding will result in decreased DNA quantity and purity. We haveobserved that grinding for only 15 min results in a 50% decrease in DNA recoveryand only 5 min results in a 75% decrease in DNA recovery compared to a 20 mingrinding effort.
3.

Transfer frozen ground plant tissue into a 50 mL conical tube and add ~10 mL pergram tissue input of the prepared ice-cold Buffer NIB (e.g., 30 mL for 3 g tissue).
• It is important to work quickly at this step – volumes do not need to be exact.
• If using a 5 g input, use caution when adding Buffer NIB to avoid letting the tube
overflow. Less than 50 mL Buffer NIB will still be sufficient for lysis.

4.

Cap the 50 mL conical tube and mix end-over-end at the maximum speed (15 rpm) for
15 min at RT (15–30°C).
• Alternatively, lay the tube horizontally on a shaker (150 rpm) or a platform rocker(maximum speed) to achieve continuous, thorough mixing.

5.

Filter the lysate using a Steriflip (20 µm pore size, 50 mL process volume).
• Using filtration methods with pore sizes larger than 20–25 µm will allow excess
debris to pass through, resulting in larger nuclei pellets that inhibit downstream lysis
and compromise DNA yield.

Quick tip
Do not filter the lysate using
pore sizes larger than 20-25µm.

• If more than 1 g tissue is being processed, more than one Steriflip may be necessary
to filter the entire volume if the filter becomes clogged.
6.

Centrifuge at the following spin speed for 20 min at 4°C.
• Use 3,000 x g for plants with genome size >1 Gb.
• Use 7,000 x g for plants with genome size <1 Gb, or if genome size is unknown.
• Note: If centrifuge cannot reach 7,000 x g, spin at the fastest speed possible.

7.

Decant the supernatant and add 1 mL of ice-cold Buffer NIB. Resuspend the pellet
using a small paint brush pre-soaked in Buffer NIB. Mix using a P1000 pipette until thesolution is homogeneous.
• Most plant species result in pellets that are too sticky to be sufficiently resuspended
by pipetting alone.

8.

Bring volume up to 15 mL with ice-cold Buffer NIB. Centrifuge at 4°C for 10 minutes
using the same centrifugation speed used in step 6.

9.

Repeat steps 7 and 8 twice. The supernatant should be clear in color after the lastcentrifugation.

© 2024 PacBio. All rights reserved. Research use only. Not for use in diagnostic procedures.
102-574-800 REV03 JUN2024

Quick tip
We recommend using a paintbrush in step 7 to resuspend
the pellet. Pipetting alone isinsufficient.

Page 7

• If the supernatant is not clear in color after 3 washes in Buffer NIB, repeat steps 7
and 8 until the supernatant is colorless.
• The number of washes is species-dependent and depends on how well the tissue
grinding was performed.
10. Decant the supernatant and add 1 mL of ice-cold Buffer 1X HB per gram of tissue
being processed (e.g. 3 mL Buffer 1X HB for 3 g tissue).

Quick tip

11. Resuspend the pellet using a small paint brush pre-soaked in ice-cold Buffer 1X HB(the paint brush from step 7 may be reused). Mix with a P1000 pipette until thesolution is homogeneous.

The nuclei pellet must be
thoroughly resuspended inBuffer 1X HB to ensure properamounts of nuclei aredistributed to each pellet.

12. Transfer 1 mL of the nuclei suspension per 1.5 mL Protein LoBind microcentrifuge
tube (equivalent to 1 g tissue/tube).

Quick tip

13. Centrifuge the 1.5 mL tubes at 7000 x g for 5 min at RT, discard the supernatant, andproceed by either snap freezing the nuclei pellet or immediately starting DNAExtraction.
• If snap freezing, place tube in liquid nitrogen to rapidly freeze the nuclei pellet andimmediately store at-80°C.
• If continuing to Nanobind DNA extraction, proceed with step 1 of the Procedure &checklist – Extracting HMW DNA from plant nuclei using the Nanobind PanDNA kit.

© 2024 PacBio. All rights reserved. Research use only. Not for use in diagnostic procedures.
102-574-800 REV03 JUN2024The Protein LoBind tubes will
improve UV 260/230 ratios by
up to 0.1–0.4 by preventing
carryover of contaminants
stuck to the tube surfaces

Page 8Results
Images of 1 g nuclei pellets are shown below:
Maize

Banana Leaf

LavenderImages of nuclei pellets after the final wash in Buffer 1X HB.

Revision history (description)

Version

Date

Initial release

01

July 2022

Minor updates

02

December 2022

Minor updates to correct extraction protocol link

03

June 2024

Research use only. Not for use in diagnostic procedures. © 2024 Pacific Biosciences of California, Inc. (“PacBio”). All rights reserved.
Information in this document is subject to change without notice. PacBio assumes no responsibility for any errors or omissions in thisdocument. Certain notices, terms, conditions and/or use restrictions may pertain to your use of PacBio products and/or third-partyproducts. Refer to the applicable PacBio terms and conditions of sale and to the applicable license terms at pacb.com/license. Pacific
Biosciences, the PacBio logo, PacBio, Circulomics, Omniome, SMRT, SMRTbell, Iso-Seq, Sequel, Nanobind, SBB, Revio, Onso, Apton,Kinnex, and PureTarget are trademarks of PacBio.
© 2024 PacBio. All rights reserved. Research use only. Not for use in diagnostic procedures.
102-574-800 REV03 JUN2024