TITLE: Spin column TNA extraction from plants - GITC method
AUTHORS: James JN Kitson

[DESCRIPTION]

This protocol is designed for extracting total nucleic acids (TNA) from plant material. In reality the drying step probably means that you won't isolate plant mRNA but viral RNA (and probably plant ribosomal RNA) are recovered. This protocol is based on the plant tissue TNA extraction method from bomb.bio but modified to use inexpensive 96 well spin column plates. The reasons for this are three-fold:

SPRI bead protocols are complicated to perform for multiple plates simultaneously. Using this protocol, up to four plates of extractions can be performed simultaneously in a deep well plate centrifuge such as the Heraeus Megafuge 40R. 
SPRI beads require very clean supernatant after grinding to prevent plant particles binding within the bead pellet and causing downstream contamination whereas the spin columns and filter plates mechanically filter the lysate.
Spin columns allow a larger amount of input material than the bomb.bio protocol.

This protocol is significantly improved through the use of high volume multichannel electronic repeaters (e.g. a Gilson PIPETMAN M P8X1200M) or a 96 well pipette (e.g. the Gilson Platemaster) but can be performed using manual multichannel pipettes.

[GUIDELINES]
Note on centrifugation in this protocol:
Maximum x g throughout is determined by both the centrifuge and the tolerances of the plastic labware you are using. If you find that not all of the liquid is moving through the spin column or that debris pellets are not pelleting, you can increase centrifugation times without any detriment to the process. Similarly, if you find plastic labware is breaking during centrifugation (this can happen with certain 0.5 ml deepwell plates), reduce the speed/g of the centrifuge and increase the centrifugation time to compensate.

Note on blocked spin columns:
Large samples and gelatinous samples can occasionally block the spin column. This can be dealt with by:
Increasing centrifuge times.
Decreasing input material.
Increasing the centrifugation time in the debris pelleting stage.

[STEPS]
SECTION: Initial digestion of OPM larvae
5. Incubate at 37 °C overnight (12-16 hours)

37 °C 
SECTION: Initial digestion of OPM larvae
3. Centrifuge at 4,000 x g for 2 min.
SECTION: DNA extraction: purification
11. Add all of the sample solution (~ 600 μL) to a well in a 96-well silica membrane spin column (we use SD5005 from NBS Biologicals) 
 

SECTION: DNA extraction: purification
12. Centrifuge at ≥ 6000 x g for 1 minute and discard the flow-through.


SECTION: DNA extraction: purification
13. Add 500 μL Wash Buffer 1 to each spin column.
SECTION: DNA extraction: purification
13.1. Wash Buffer 1 should be comprised of the following reagents:

 

SECTION: DNA extraction: purification
14. Centrifuge at ≥ 6000 x g for 1 minute and discard the flow-through.


SECTION: DNA extraction: purification
15. Add 500 μL Wash Buffer 2 to each spin column.
SECTION: DNA extraction: purification
15.1. Wash Buffer 2 should be pH ~7 and comprised of the following reagents:

 

SECTION: DNA extraction: purification
16. Centrifuge at 20,000 x g for 3 minutes.


SECTION: DNA extraction: purification
17. Discard the collection tube and replace it with a new 1.5 mL microcentrifuge tube.
SECTION: DNA extraction: purification
18. Add 100 - 200 μL Elution Buffer directly to the silica membrane and leave it at room temperature for 5 minutes.
SECTION: DNA extraction: purification
18.1. Elution Buffer should be pH ~7 and comprised of the following reagents:
 
 

SECTION: DNA extraction: purification
19. Centrifuge at ≥ 6000 x g for 1 minute. The DNA is now in the collection tube and can be taken forward to amplification. 
 

SECTION: Initial digestion of OPM larvae
2. Grind the larvae is a tissue homogeniser until homogenised.

 

SECTION: Initial digestion of OPM larvae
4. To the lysate, add 1 mL of freshly-prepared Proteinase Buffer, a master mix of Lysis Buffer 2 and Proteinase K (detailed in the sub-steps below) and vortex to mix.
 

SECTION: Initial digestion of OPM larvae
4.1. Lysis Buffer 2 should be pH 9 and comprised of the following reagents:
 
 

SECTION: Initial digestion of OPM larvae
4.2. Per sample, the Proteinase Buffer master mix should comprise:

 

SECTION: Initial digestion of OPM larvae
6. Centrifuge at 4000 x g for 4 minutes.
SECTION: Initial digestion of OPM larvae
7. Transfer 1.5 mL of the supernatant to a clean 2 mL screw-cap tube for archiving/backup.
SECTION: Initial digestion of OPM larvae
8. Centrifuge at 10,000 x g for 1 minute.
SECTION: Initial digestion of OPM larvae
9. Transfer 200 μL of the supernatant to a clean 1.5 mL microcentrifuge tube. 
 

SECTION: DNA extraction: purification
10. Add 400 μL of master mix of Protein Denaturation Buffer and ethanol (detailed below) to each sample.
SECTION: DNA extraction: purification
10.1. Protein Denaturation Buffer should be comprised of the following reagents:
  
 

SECTION: DNA extraction: purification
10.2. Per 96-well plate, the Protein Denaturation Buffer and ethanol master mix should comprise:

 

SECTION: DNA extraction: purification
None. Protein Denaturation Buffer should be comprised of the following reagents:
  
 

SECTION: DNA extraction: purification
None. Add 200 μL of the supernatant from STEP 10 into each well containing Protein Denaturation Buffer and ethanol. 
SECTION: DNA extraction: purification
None. Per 96-well plate, the Protein Denaturation Buffer and ethanol master mix should comprise:

 

SECTION: DNA extraction: purification
None. Add all of the sample solution (~ 600 μL) to a well in a 96-well DNA membrane spin column and cover with a breathable seal. 
 

SECTION: DNA extraction: purification
None. Centrifuge at ≥ 6000 x g for 1 minute and discard the flow-through.
 

SECTION: DNA extraction: purification
None. Add 500 μL Wash Buffer 1 to each spin column and cover with a breathable seal.
SECTION: DNA extraction: purification
None. Centrifuge at ≥ 6000 x g for 1 minute and discard the flow-through.
 

SECTION: DNA extraction: purification
None. Wash Buffer 1 should be comprised of the following reagents:

 

SECTION: DNA extraction: purification
None. Add 500 μL Wash Buffer 2 to each spin column and cover with a breathable seal.
SECTION: DNA extraction: purification
None. Wash Buffer 2 should be pH ~7 and comprised of the following reagents:

 

SECTION: DNA extraction: purification
None. Centrifuge at 20,000 x g for 3 minutes and discard the flow-through.
 

SECTION: DNA extraction: purification
None. Carefully move the spin column plate to a new 0.5 mL DNA collection plate.
 

SECTION: DNA extraction: purification
None. Add 200 μL Elution Buffer directly to the silica membrane and leave it at room temperature for 5 minutes, covering with a breathable seal.
SECTION: DNA extraction: purification
None. Elution Buffer should be pH ~7 and comprised of the following reagents:
 
 

SECTION: DNA extraction: purification
None. Centrifuge at ≥ 6000 x g for 1 minute. The DNA is now in the collection plate and can be taken forward to amplification. 
 

SECTION: Initial digestion of OPM larvae
1. When ready to begin tissue digestion, defrost the tubes containing dead larvae in Lysis Buffer 1.
SECTION: Preparation of plant material
3. Prepare TNES buffer: 
    ABC1ReagentRequired cencentration in bufferAmount per litre2CTAB2%20 g3Polyvinyl pyrrolidone2%20 g4Tris-HCl10 mM10 ml (of 1M stock solution)5EDTA20 mM40 ml (of 0.5M stock solution6Ammonium chloride1.3M69.54 g

 

 
 
SECTION: Preparation of plant material
2. Beads are usually shipped coated in manufacturing oil (especially the carbon steel beads). To remove this, place beads in a borosilicate glass beaker or Duran bottle with the pouring lip and lid removed then bake for at least 12 hours at 250 oC. 

  

 

SECTION: Preparation of plant material
1. Source steel beads (ball bearings) for tissue grinding (Tungsten beads are not usually necessary). We use hardened carbon steel or stainless steel bearings from simplybearings.co.uk. This protocol requires three beads per sample tube.
SECTION: Preparation of plant material
4. Prepare 5 ml screwcap collection tubes (e.g. Eppendorf Cat. No. 0030122305) containing three 3 mm hardened steel beads in batches of 92 tubes.

SECTION: Preparation of plant material
5. Using forceps (either disposable forceps or ethanol and flame steel forceps between samples), place ~ 50 - 100 mg of wet plant material into the pre-prepared tubes containing the hardened steel ball bearings. 

SECTION: Preparation of plant material
6. Place plates in a -80 freezer for at least 30 min before grinding. Or alternatively snap freeze in liquid nitrogen.
SECTION: Preparation of plant material
8. Place tubes while still cold in tissue homogeniser and grind the plant material in a tissue homogeniser until homogenised.

 

SECTION: Preparation of plant material
9. To the plant material, add 500 μl of freshly-prepared TNES, and place in a tube shaker for 5 minutes or tissue homogeniser (at 1750 RPM for one minute) to mix.
 

SECTION: Preparation of plant material
11. Centrifuge at maximum x g for 10 minutes to pellet plant debris.

 

SECTION: Preparation of plant material
7. 
Everything from this point forward needs to be performed in pairs of plates so that your centrifuge is balanced.
SECTION: Preparation of plant material
12. Arrange 5 ml tubes in a 96-well format according to a predefined sample order (or randomly and record tube order). Starlab 5ml tube racks are useful for this. 3D printed options exist as well. 
 

  

SECTION: Preparation of plant material
13. Transfer the supernatant (approximately 400 μl) to a new 96 well filter plate (NBS biologicals #SD5006) placed on top of a 0.5 ml deep well plate (Sarstedt #82.1969.002 or similar). Seal the filter plate with a breathable plate seal (Starlab #E2796-3005). Make sure you record the location of each sample on the plate.

 
SECTION: Preparation of plant material
14. Place both filter plate and deepwell plate in centrifuge at maximum x g for 5 minutes. 



SECTION: Preparation of plant material
15. Transfer 60 μL of the supernatant to a clean 1.2 mL deep-well 96-well plate. Seal the 0.5ml plate with a non-breathable plate seal or caps.
 

SECTION: Preparation of plant material
10. 
 

SECTION: Preparation of plant material
16. Add 120 μL 1.5X GITC buffer (detailed in the sub-step below) and 240 μL of Isopropanol to each well of the 1.2 mL 96-well plate and mix by pipetting or seal and shake on a plate shaker.
SECTION: Preparation of plant material
16.1. 1.5X GITC should be comprised of the following reagents:
  
 

SECTION: Preparation of plant material
17. Add all of the sample solution (~ 420 μL) to a well in a 96-well silica membrane spin column (we use SD5007 from NBS Biologicals) and cover with a breathable seal. 
 

  

SECTION: Preparation of plant material
18. Leave for 5 min then centrifuge at maximum speed for 10 minutes.
 

SECTION: Preparation of plant material
19. Add 400 μL Isopropanol to each spin column, cover with a breathable seal and leave for two minutes.
SECTION: Preparation of plant material
20. Centrifuge at maximum speed for 5 minutes and discard the flow-through.


SECTION: Preparation of plant material
21. Wash spin columns three times as in substeps below with 80% Ethanol solution.
SECTION: Preparation of plant material
21.1. Add 300 μL of 80% Ethanol solution to each well and centrifuge at maximum speed for 10 minutes.

SECTION: Preparation of plant material
22. Centrifuge spin columns for two further minutes at full speed to remove any last traces of ethanol from the silica membranes.
 

SECTION: Preparation of plant material
23. Carefully move the spin column plate to a new 0.5 mL DNA collection plate.


SECTION: Preparation of plant material
24. Add 100 μL Elution Buffer or nuclease free water directly to the silica membrane and leave it at room temperature for 5 minutes, covering with a breathable seal.
SECTION: Preparation of plant material
24.1. Elution Buffer should be pH ~7 and comprised of the following reagents:
 
 

SECTION: Preparation of plant material
25. Centrifuge at ≥ 6000 x g for 2 minutes. The DNA is now in the collection plate and can be taken forward to amplification.