TITLE: 96 well spin column TNA extraction from plants - CTAB method
AUTHORS: James JN 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 patent US 20110092687 A1 to allow the use of CTAB with spin columns instead of the more usual chloroform/Isoamyl alcohol method.

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 tissue debris is 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: Preparation of grinding tubes and buffer
1. Source steel beads (ball bearings) for tissue grinding (Tungsten beads are not usually necessary). We use hardened 3mm carbon steel or stainless steel bearings from simplybearings.co.uk. This protocol requires three beads per sample tube.
SECTION: Initial homogenisation of plant material
7. Place tubes while still frozen in tissue homogeniser and grind the plant material in a tissue homogeniser until homogenised.

 

SECTION: Initial homogenisation of plant material
8. To the plant material, add freshly-prepared CTAB lysis buffer (see below for volumes), and place in a tube shaker, vortex mixer or tissue homogeniser (at low speed) to mix.
 

 

SECTION: Initial homogenisation of plant material
10. Centrifuge at 1500 x g for 5 minutes to pellet plant debris.


 

SECTION: Preparation of grinding tubes and buffer
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 grinding tubes and buffer
3. Prepare CTAB lysis buffer:

 

SECTION: Initial homogenisation of plant material
9. Incubate lysed samples in CTAB lysis buffer.

55 °C 

SECTION: Preparation of grinding tubes and buffer
4. Prepare 5 ml preparation tubes (e.g. Starlab #E1450-1100) containing three 3 mm hardened steel beads in batches of 90 tubes.
SECTION: Preparation of plant material
5. Using forceps (either disposable forceps or ethanol and flame steel forceps between samples), place ~ 50 mg of dried plant material (100 mg if using fresh) into the pre-prepared collection tubes containing the hardened steel ball bearings. 
 


SECTION: Preparation of plant material
6. Place all sample tubes in zip-lock bags in batches of 90 (to allow for two extraction negatives, a DNA negative and three positive controls). 

Freeze at -80oC until ready to proceed with the extraction.
SECTION: Initial homogenisation of plant material
11. Transfer 350 μl of supernatant to a new 96 well filter plate (NBS biologicals #SD5006) placed on top of a 1-2 ml deep well plate. Seal the filter plate with a breathable plate seal (Starlab #E2796-3005)

Add 350 μl of CTAB buffer to two of your empty wells designated as extraction negatives.
SECTION: TNA purification
13. Add 350 μl of 100% Isopropanol to each sample and vortex to mix.
SECTION: TNA purification
14. Centrifuge at 1500 x g for 5 minutes to pellet plant debris.
 

SECTION: TNA purification
15. For each sample, add as much as possible* of the supernatant from step 14 to a well in a 96-well silica membrane spin column (we use SD5007 from NBS Biologicals) sitting on top of a 1-2 ml deep well plate and cover with a breathable seal. 


 

SECTION: TNA purification
17. Discard flow-through or change waste receiver and add 500 μl of 70% Ethanol. Centrifuge at 1500 x g for 5 minutes.
SECTION: TNA purification
18. Repeat step 15 two more times for a total of three washes. After the last wash spin for 10 minutes to dry the spin column.
SECTION: TNA purification
19. Transfer the spin column to a new 0.5 ml deep well plate and add 50-100 μl of water or 10mM Tris-HCl. Incubate for 10 minutes at room temperature and centrifuge at 1500 x g for 1 minute.

The flow-through now contains the extracted TNA.
SECTION: Preparation of grinding tubes and buffer
4. Prepare 5 ml screwcap collection tubes (e.g. Starlab #E1450-1100) containing three 3 mm hardened steel beads in batches of 96 tubes.
SECTION: TNA purification
5. Using forceps (either disposable forceps or ethanol and flame steel forceps between samples), place ~ 50 mg of dry plant material (100mg of fresh material) into the pre-prepared collection tubes containing the hardened steel ball bearings. 
 



SECTION: TNA purification
6. Place all sample tubes from a session in a zip-lock bag and freeze at -80oC until ready to proceed with the extraction.
SECTION: Preparation of grinding tubes and buffer
7. Place tubes while still frozen in tissue homogeniser and grind the plant material in a tissue homogeniser until homogenised.

 

 

SECTION: Preparation of grinding tubes and buffer
8. To the plant material, add freshly-prepared CTAB lysis buffer (see below for volumes), and mix thoroughly on a tube shaker, vortex mixer or in the tissue homogeniser at low speed.
 

 

SECTION: Preparation of grinding tubes and buffer
9. Incubate lysed samples in CTAB lysis buffer.

55 °C 

SECTION: Preparation of grinding tubes and buffer
10. Centrifuge at 1500 x g for 5 minutes to pellet plant debris.


 

SECTION: Preparation of grinding tubes and buffer
11. Take 350 μl of supernatant from each sample and pipette it into an empty well on a filter plate siting on top of a 1 - 2 ml deep well plate.

Record the location of that sample for later reference.
SECTION: TNA purification
13. Add 350 μl of 100% Isopropanol to each sample and vortex to mix.
SECTION: TNA purification
14. Centrifuge at maximum x g for 2 minutes to pellet plant debris.
 

SECTION: TNA purification
15. Add all of the supernatant from step 14  into an empty well on a 96 well spin column plate siting on top of a 1 - 2 ml deep well plate and centrifuge at 1500 x g for 5 minutes.
SECTION: TNA purification
16. Discard flow-through or change waste receiver and add 500 μl of 70% Ethanol. Centrifuge at 1500 x g for 5 minutes.
SECTION: TNA purification
17. Repeat step 15 two more times for a total of three washes. After the last wash spin for 10 minutes to dry the spin column.
SECTION: TNA purification
18. Transfer the spin column to a new 0.5ml deep well plate and add 50-100 μl of water or 10mM Tris-HCl. Incubate for 5 minutes at room temperature and centrifuge at 1500 x g for 1 minute.

The flow-through now contains the extracted TNA.
SECTION: Preparation of grinding tubes and buffer
12. Centrifuge at 1500 x g for 1 minute to fragment and filter any remaining plant material
SECTION: Initial homogenisation of plant material
12. Centrifuge at 1500 x g for 1 minute to filter and further homogenise the plant material.
 

SECTION: TNA purification
16. Centrifuge at 1500 x g for 5 minutes.