plant surface microbiology.pdf

29 Functional Genomic Approaches for Studies of Mycorrhizal Symbiosis 569
Boekel cooler or similar to maintain 16 °C temperature
Gel electrophoresis equipment and power supplies
Hybridization oven (Amersham or Fisher biotech or similar)
Variable volume pipettes
High resolution scanner with transparency adapter
Phosphorimager (Bio-Rad Personal Imager FX)
2.2 Biological Material
Biological material used for the RNA extraction is collected as quickly as possible,
immediately frozen in liquid nitrogen and stored at –80 °C.
2.3 RNA Extraction
The protocol is modified from the one described by Chomoczynski and Sacchi
(1987).
Reagents
Extraction buffer:
4 M Guanidinium thiocyanate
25 mM Na-citrate pH 7
0.5 % Na-laurylsarcosine
0.1 M b-mercaptoethanol (added just prior to use)
2 M Na-acetate pH 4
Phenol water-saturated
Chloroform:isoamylalcohol (49:1, v/v)
Isopropanol
8M LiCl
80 % ethanol
Procedure
Grind the material (100 mg) in liquid nitrogen (with a pestle in a microfuge
tube or in a mortar)
1. Add 200 ml of extraction buffer and place on ice.
2. Add 40 ml of 2 M Na-acetate pH 4, mix thoroughly by inversion.
3. Add 400 ml of phenol and mix by inversion.
4. Add 150 ml of chloroform/isoamylalcohol, mix by inversion and place on
ice 10 min. Centrifuge at 10,000 g for 20 min at 4 °C.
5. Transfer the aqueous phase into a new tube and extract with an equal volume
of chloroform/isoamylalcohol.
6. Transfer the aqueous phase into a new tube and add 1 volume of isopropanol.