plant surface microbiology.pdf

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Gopi K. Podila and Luisa Lanfranco
7. Incubate 2 h at –20 °C.
8. Centrifuge at 10,000 g for 20 min at 4 °C. Remove the supernatant.
9. Wash the pellet with 80 % ethanol.
10. Resuspend in 50–100 ml of sterile water and store at –80 °C.
Note: As an alternative to the RNA extraction protocol explained above, commercial
kits from several companies are available. These usually have no need
of phenol:chloroform manipulations and are relatively rapid. RNA obtained
with these kits often needs to be treated with RNase-free DNase to remove DNA.
DNase Treatment
Incubate the RNA sample in DNase 1¥ buffer (100 mM Tris-HCl pH 7.5,
10 mM MgCl 2 , 1 % BSA) with units of DNase (RNase-free; Promega, Madison,
WI, USA) for 30 min at 37 °C.Add EDTA for 2 mM final concentration. Extract
with an equal volume of phenol/chloroform/isoamylalcohol (25/24/1; v/v/v).
Precipitate the RNA with Na-acetate (0.3 M final concentration) and ethanol
(2.5 volumes). As an alternative, to remove contaminant DNA, a precipitation
with LiCl (final concentration 2 M, overnight at 4 °C) can be performed.
3 RNA Quantification
RNA quantification can be determined with a spectrophotometer (A 260/280)
or fluorometer (Amersham Pharmacia Biotech). The quality of RNA should
be checked on a denaturing agarose gel (Sambrook and Russel 2001) to make
sure that the integrity of RNA is good.
3.1 Construction of a cDNA Library
There are many kits available for the construction of a cDNA library. If there
is plenty of total RNA available to purify poly-A RNA, standard cDNA synthesis
kits can be used such as lambda zap kits (Stratagene, CA, USA). However,
if the availability of the amounts of RNA is limited, it is advisable to use a kit
that can use either a small amount of total RNA or poly-A RNA to synthesize
the cDNA library. Because the amount of tissue and RNA available from mycorrhizal
tissues or mycorrhizal fungi is often limited, we describe here the
method of synthesizing a cDNA library using the SMART cDNA library construction
kit (Clontech, CA, USA). This kit can work on as little as 50 ng of
total RNA since it uses an amplification step after the first strand cDNA synthesis
that compensates for small amounts of starting RNA material.