TECHNIQUES IN PLANT VIROLOGY

CIP Training Manual

2.4 DETECTION/PSTVd

Section 2.4.2
Detection of PSTVd by
Polyacrylamide Gel
Electrophoresis (PAGE)

Until recently, the electrophoretic separation in acrylamide gels of low

molecular weight RNAs isolated from plants was widely used for
detecting the spindle tuber viroid of potato. This method, along with the
tomato test, can be used in routine work for detecting PSTVd.

1. Preparation of stock solutions

a) EDTA 0.1 M

Dissolve 33.62 g EDTA disodium salt in 1 liter of distilled water.

b) Ammonium hydroxide 4 M
 Add distilled water to 140 ml NH4OH to make 1 liter final volume.
Mix well.

c) Lithium chloride 10 M

Dissolve 423.9 g LiCl in 1 liter of distilled water (final volume).

2. Sample preparation

Work at low temperature (on ice). Prepare the extraction buffer

solution:

60 ml LiCl (10 M)
20 ml EDTA (0.1M)
20 ml NH4OH (4M)
60 ml distilled water

Use 1.5 ml of extraction buffer solution for each 0.5 g sample.

Macerate and extract sap. Place it in an Eppendorf tube. Add an
equal amount of saturated phenol and mix in a vortex.

Centrifuge at 14,000 rpm for 5 minutes. Recover the aqueous

phase (top) and add 2-1/2 volumes of absolute ethanol. Store
overnight at –20 OC in a freezer.

On the following day, centrifuge at 10,000 rpm for 10 minutes and

resuspend sediment in 2 drops of phosphate buffer solution 0.067
M, pH 7.2.

3. Preparation of acrylamide gel

The preparation of acrylamide gel requires the following solutions:

a) Electrophoresis buffer solution (5X):

TRIS base 0.18M 10.90 g 21.8 g

Sodium Phosphate 0.15 M
(NaH2PO4 2H2O) 10.35 g 20.7 g
EDTA 0.005 M 0.73 g 1.46 g
Distilled water
a final volume of 500 ml 1000 ml

b) Acrylamide solution:

15% acrylamide
(specially purified) 15 g 150 g
0,75% NN-Methylene
bisacrylamide (purified) 0.75 g 7.5 g
Distilled water to 100 ml 1000 ml

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 c) Fresh 10% w/v ammonium persulfate solution. Dissolve 1.25 g
ammonium persulfate in distilled water to a final volume of 12.5 ml.

Mix solution for the gel in a 250-ml Erlenmeyer flask. The

concentration of acrylamide gel used for PSTVd detection is 5%.
Add 30 ml (5X) electrophoresis buffer solution and 67 ml distilled
water for every 50 ml 15% acrylamide solution.

To this solution, add 0.25 ml TEMED (catalyzer) and 2.5 ml of

fresh 10% ammonium persulfate solution. Stir gently for a few
seconds.

Pour mix in the electrophoresis tank and insert comb. Wait 5 to 10

minutes until gel polymerizes.

Once the gel is polymerized, remove the comb. Add

electrophoresis buffer solution 1X to both tanks (upper and lower).
Pre-run for 30–60 minutes at 100 V.

4. Adding samples

To each sample, add 1 drop glycerol (or 2 drops 50% sucrose in

electrophoresis buffer solution) and 1 drop bromophenol blue (at 1% in
electrophoresis buffer solution). Mix well.

Use a Pasteur pipette or a micropipette to place each sample carefully in

the wells made by the comb.

Run gel at 100 V until the front of the bromophenol blue is 0.5 cm from
the end of gel.

5. Gel staining

a) Staining with Toluidine blue.

Dissolve 0.03 g Toluidine blue in 300 ml distilled water. Pour dye in

the deposit containing the gel and shake gently over night. The
next day, wash gel several times in water until the bands can be
seen. Read results on a light transilluminator.

b) Staining with ethidium bromide

Wear gloves. Prepare a 10 mg/ml ethidium bromide stock solution.

In a glass petri dish, large enough to contain the gel, mix 300 ml
distilled water and 60 l ethidium bromide stock solution. Carefully
place gel in the staining solution. Stain for 10 to 15 minutes and
read results on an ultraviolet light transilluminator.

To decontaminate ethidium bromide solution: add 100 mg powder

activated charcoal to every 100-ml ethidium bromide solution. Mix
at room temperature for 1 hour, stirring occasionally. Filter with
Whatman No. 1 filter paper and eliminate liquid. Place filter paper
with activated charcoal in a sealed plastic bag and dispose of in an
adequate container.

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 NOTE: Use this method only with thin gels (2 mm or less).

c) Staining with silver nitrate.

Wear gloves throughout the process. Prepare the following

solutions:

• Silver nitrate (340 mg in 200 ml distilled water).

• Ethanol 10% (50 ml absolute ethanol in 450 ml distilled water).
• Acetic acid 0.5% (2.5% acetic acid in 500 ml distilled water final
volume).
• Developer:

3 g sodium hydroxide (NaOH)

17 mg sodium borohydride (NaBH4)
0.8 ml formaldehyde 37% (HCHO)

• A solution of sodium carbonate (1.48 g CO3Na2 brought to a final

volume of 200 ml with sterilized distilled water).

For development, wash gel twice for 5 minutes. Each time wash
first with 10% ethanol and then with silver nitrate for 15 minutes.
Rinse with distilled water 4 to 5 times for 15 seconds.

Develop for 1 to 5 minutes (at most) with developer. Stop reaction

by immersing gel in distilled water for 5 minutes.

6. Preparation of saturated phenol

a Using liquid phenol

NOTE: This complete procedure should take place under an air

extraction fan. Wear thick gloves and mask. Phenol is toxic and
may cause burns. Wear special goggles when mixing.

Add 0.1% 8-hydroxyquinoline and 10% m-cresol to liquid phenol and

stir for one hour.

To stabilize pH, wash 3 to 4 times (add buffer solution; mix; wait

for phase separation; and dispose of solution) with Tris-HCl (1 M)
buffer solution pH 8.0 and then with 0.1 Tris-HCl pH 8.0 until the
pH of the phenol solution is above 7.6.

Store at 4 oC. Under these conditions, the solution is stable for 1 to

2 months.

b) Using crystallized phenol (for 500 g)

Add 140 ml distilled water to flask with phenol. Once dissolved,

add 10% m-cresol and stir for 1 hour.

Add 0.68 g 8-hydroxyquinoline and stir again for 1 hour.

Leave at room temperature overnight. The next day, stabilize pH

as with liquid phenol.
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 Recommended Literature
Martínez, E. 1984. La tinción de geles en electroforesis con nitrato de
plata y su aplicación para identificar viroides. Tesis Biol. Univ. Nac.
Pedro Ruiz Gallo, Lambayeque, Perú. 39 pp.
Morris, T.J. and N.S. Wright. 1975. Detection on polyacrylamide gel of a
diagnostic nucleic acid from tissue infected with potato spindle tuber
viroid. American Potato Journal 52:57.

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