2.5.2.1. Preparation of LB Media Reagents:
LB (Luria-Bertani) broth [1% (w/v) bacto-tryptone (DIFCO Laboratories, Detroit, MI, USA), 0.5% (w/v) bacto-yeast extract (DIFCO Laboratories), 1% (w/v) NaCl, pH 7.0]
Amp100 (Ampicillin 100 mg/mL)
1.5% (w/v) agar (Life Technologies, Gathersbur, MD, USA)
Glycerol
0.1 M IPTG stock solution (filter-sterilised and aliquots stored at -20 C)
X-Gal (50 mg/ml dissolved in N,N’-dimethyl-formamaide)
E coli cultures were maintained on LB plates. LB broth growth medium, supplemented with 100 g mL-1 Ampicillin (LB-Amp100 broth), was used to grow liquid E. coli
cultures with vigorous shaking (200 rpm) at 37 C. To make the LB broth, all components of LB media were mixed by stirring, the pH was adjusted to 7.0 and this solution autoclaved. When required, 100 g mL-1 of Ampicillin was added to the
cooled media following autoclaving. To prepare the LB plate, 1.5% (w/v) agar was added to the LB media prior to autoclaving and when required, the media was supplemented with 100 g mL-1 Ampicillin (LB-Amp100 medium) or with 100 g mL-1
Ampicillin, 0.5 mM IPTG and 80 g mL-1 X-Gal (LB Amp100/IPTG/X-Gal) as
appropriate. These components were added when the agar medium had cooled down to
ca. 50 C, after which the medium was poured into sterile plates in a laminar flow cabinet, left to solidify for 15 min and plates were sealed using parafilm and kept at 4
C until required.
2.5.2.2. Preparation of SOC Medium Reagents:
2 % (w/v) bacto-tryptone (DIFCO Laboratories, Detroit, MI, USA)
0.5% (w/v) bacto-yeast extract (DIFCO Laboratories)
1% (w/v) NaCl
2.5 mM KCl
20 mM filter sterilised-Mg2+ (from stock of from 10 mM MgCl2 + 10 mM MgSO4)
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20 mM filter sterile-glucose (from stock of 1M glucose)
SOC medium was prepared by adding all components above (except the Mg2+ and glucose). Following sterilization by autoclaving, the media was left to cool and filter sterilized Mg2+ stock and glucose was added, each to the final concentration of 20 mM. The final pH was 7.0.
2.5.2.3. Preparation of Competent Cells Reagents:
60 mM CaCl2 Glycerol
LB broth
E. coli cells used for transformation were prepared from E. coli strain DH5 (GIBCO BRL). Bacterial cells, from a single colony or a glycerol stock, were cultured in 10 mL LB broth at 37 C overnight with vigorous shaking (200 rpm). An aliquot (0.5 mL) of the overnight culture was removed and used to inoculate 500 mL of fresh LB media, the culture was then incubated at 37 C until cell growth reached an optical density of 0.6 at 600 nm. The cells were pelletted by centrifugation at 6000 x g for 5 min at 4 C, the pellet resuspended in 10 mL of ice-cold 60 mM CaCl2 followed by the addition of
a further 10 mL of ice-cold 60 mM CaCl2. Following incubation on ice for 30 min, the
cell suspension was centrifuged at 2,000 x g for 5 min at 4 C and the pellet resuspended in 4 mL of 60 mM CaCl2 containing 15% (v/v) glycerol. Aliquots of the
cell suspension (300 L) were transferred to microfuge tubes, snap frozen in liquid nitrogen and stored at -80 C until required for transformation.
2.5.2.4. Preparation of Glycerol Stock Reagents:
LB Broth
Glycerol
Single colonies of interest were used to inoculate 10 mL LB broths (see 2.5.2.1) which were cultured at 37 C with vigorous shaking (200 rpm) overnight. A 850 L of
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aliquot of the overnight culture was added into a cry tube containing 150 L of glycerol, the suspension mixed, snap frozen and stored at -80 C.
2.5.3. Isolation and Quantification of RNA
2.5.3.1 Extraction of Total RNA Reagents:
Borate Buffer (200mM di-sodium tetraborate decahydrate, 500 mM EDTA, 10 % (w/v) EDTA, 10% (w/v) sodium deoxycholate, 1% (w/v) SDS, 100 mM DTT), pH 9.0
Extraction Buffer (Borate Buffer, 2% (w/v) PVP-40, 1 % (w/v) IGEPAL CA-630), pH 9.0
Proteinase-K (20 mg/mL)
2 M KCl, 4M LiCl and 3 M Sodium acetate
Chloroform: isoamyl-alcohol (24:1 (v/v))
Isopropanol
80% (v/v) ethanol
DEPC-treated water
Total RNA was isolated using the hot borate method (Hunter and Reid, 2001; Moser et al., 2004) with some modifications. To prevent RNA degradation during extraction, all glassware, mortars and pestles, and spatulas used were wrapped with aluminum foil and baked in a dry oven at 180 C for at least 14 h. All disposable plastic was either new, or treated overnight in 0.3% (v/v) hydrogen peroxide (Andrew Industrial Ltd), then rinsed well with Milli-Q water, wrapped with aluminium foil and sterilised by autoclaving. Chemicals used for RNA work were not used for other purposes and weighed by pouring the chemicals. Clean disposable gloves were always used and regularly changed. All solutions used were made to required concentrations by DEPC- treated water.
To extract total RNA from leaf samples, frozen tissues (100 mg for apical structures and 200 mg for first and second fully-expanded leaves) were ground to a fine powder in liquid nitrogen using a pre-chilled mortar and pestle before transfer into a microtube containing five volumes (w/v) of warm (85 C) extraction buffer. The tissues and extraction buffer were mixed by vortexing for 30 sec, and before Proteinase-K (0.75 %; v/v) was added to the slurry. The tubes containing the mixtures were incubated at 42 C, with shaking at ca. 100 rpm, for 90 min. Immediately after
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incubation, 2 M KCl was added to a total concentration of 160 mM [0.08 (v/v)], mixed and incubated in an ice bath and shaken horizontally at ca. 100 rpm for 30 min. The extraction mix was then centrifuged at 10 800 x g for 20 min at 4 C, and the supernatants transferred to a fresh tube and an equal volume of cold 4 M LiCl (to give final concentration of 2 M) added and after mixing well, the RNA was precipitated by overnight incubation at 4 C. The precipitate was then collected by centrifugation at 20 8000 x g for 30 min at 4 C, the supernatant discarded and the pellet resuspended in 500 l of DEPC-treated water prior to the addition of 50 L of 3 M sodium acetate (to give a final concentration 0.3 M) and 550 L [1: 1 (v/v)] of chloroform/isoamyalcohol. The aqueous and organic phases were shaken vigorously for 30 sec and then separated by centrifugation at 20 800 x g for 5 min at room temperature. The upper aqueous phase was carefully pipetted and transferred into a fresh sterile microfuge tube and then isopropanol (1:1; v/v) was added, the contents mixed well by inverting the tube and then the mixture incubated on ice for 1 h or at 4 C overnight to precipitate the RNA. The RNA was then pelletted by centrifugation at 20 800 x g for 30 min at 4 C, the pellet washed with 500 L of 80 % (v/v) ice-cold ethanol, air dried for 5 min and resuspended in 500 L of DEPC-water. To remove genomic DNA contamination, the RNA was routinely precipitated with 4 M LiCl (added to a final concentration of 2 M) and incubated either overnight at 4 C or 1 h on ice. The RNA was pelletted by centrifugation at 20 800 x g for 30 min at 4 C, then washed with ice-cold 80% (v/v) ethanol, air dried, then resuspended in 30 to 50 L of DEPC-water. The RNA was quantified (Section 2.5.3.2), and aliquots (5 L) snap frozen in liquid nitrogen and stored at -80 C until required.
2.5.3.2. Quantification of RNA in Solution
The total RNA concentration was determined by measuring the absorbance at 260 nm (A260) using an Ultrospec uv/visible spectrophotometer (Pharmacia Biotech). Samples
were diluted and aliquots of 100 L were transferred into a quartz cuvette and measured against a DEPC-water blank. For RNA, an OD260 of 0.1 corresponds to
approximately 40 µg mL-1 (Sambrook et al., 1989). Therefore, the equation to determine RNA concentration is:
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The purity of RNA was determined by measuring the A260/A280 ratio (also measured
against a water blank), with relatively pure RNA solutions have an A260/A280 ratio of
2.0 (Sambrook et al., 1989).