2.8.1 Synthesis of single-stranded cDNA
Leaf poly A ^ RNA (2 fig) and 0.4 fig oligo dT,2 . , 8 primers were mixed in a
final volume of 17 fil, denatured at 65°C for 10 min and annealed at room temperature for 10 min. The cDNA synthesis reaction was set up containing the annealed RNA-primers, 0.2 mM dATP, 1 mM dCTP, 1 mM dGTP, 1 mM TTP, 50 mM Tris-HCl pH 8.3, 50 mM KCl, 8 mM MgCl^, 5 mM DTT, 1 ^Ci p'P]-
dATP, 1.25 U RNasin (Amersham), and 30 U AMV reverse transcriptase (N.B.L. Life Sciences). This was incubated at 42“C for 60 min, following which 2 /xl were removed for scintillation counting and the remainder ethanol precipitated and resuspended in 50 fil TE. The 2 fil reserved for scintillation counting was spotted onto a Whatman DE81 filter, washed 6 times with 0.5 M NagHPO^, twice with
SDW and twice with 95% (v/v) ethanol. The filter was then dried, placed in Cocktail ’O’ and counted.
2.8.2 Oligonucleotide preparation
Oligonucleotides B (' CAC ATT GCT GCA GAT GGA GAA CP") and C C’ACA TAT GGA TCC ATG TTA GAA GCA CP") were designed from two highly conserved regions of plant GS genes, that flank intron 11. For cloning purposes, oligonucleotide B was designed to contain a Pstl site while
oligonucleotide C was designed to contain a Baml^X site. The oligonucleotides were purified by a modified procedure of Sawadogo and Van Dyke (1991) whereby 85 /zl of NH4OH (35% NH3) was vortexed for 15 s with 25 /d
oligonucleotide solution and 1.1 ml butan-l-ol. After centrifugation at 15000 x g for 1 min the pellet was resuspended in 1(X) /d SDW and a further 1 ml butan-l-ol added, vortexed, and spun. The pellet was finally redissolved in 100 /xl SDW and the concentration adjusted to 5 /xg./d'\
2.8.3 ’Hot start’ PCR
The PCR reaction was set up using the GeneAmp PCR kit (Perkin Elmer Cetus) whereby 0.6 pmol./xl^ of each primer, 0.2 mM dATP, 0.2 mM dCTP, 0.2 mM dGTP, 0.2 mM TTP, 10 mM Tris-HCl pH 8.3, 50 mM KCl, 1.5 mM MgCl^, and 0.001% gelatin were added to the target DNA in a final volume of 50 /d. This was overlaid with 50 /d mineral oil and heated to 94°C for 2 min prior to addition of 1 U AmpliTaq DNA polymerase (Perkin Elmer Cetus). The annealing and elongation temperatures and the number of cycles for each amplification are stated in the results Chapter. The most suitable annealing temperatures were calculated by using the equations published by Breslauer et al. (1986).
Upon completion 100 /d chloroform was added to remove the mineral oil and the aqueous phase recovered. The PCR product was ethanol precipitated and resuspended in 10 /xl TE.
2.9.1 Ligation
The products of the PCR reaction were digested with Pst\ (Gibco BRL) and BamRl (Boehringer Mannheim), and the DNA fragments purified using the Prep- A-Gene kit (Chapter 2.7.1) and ligated in 50 mM Tris-HCl pH 7.6, 10 mM MgCla, 5% (w/v) polyethylene glycol 80(X), 1 mM ATP, 1 mM DTT, 25 ng pUC9 (cut with Pstl and BarnRl), 25 ng target DNA, and 1 U T4 DNA ligase (Pharmacia) in a total volume of 5 /^l. The reaction was incubated overnight at 14°C, 45 jul of TE was added and it was stored at -2frC.
2.9.2 Transformation
Aliquots (1 fA and 5 fi\) of the ligated DNA were diluted to 25 /xl. The vector pUC9 (50 pg) was also diluted to 25 fi\ and used a negative control. 50 fil aliquots of competent E. coli cells (from J. Boxall) were transferred to the diluted ligation mix and this was placed on ice for 30 min. The samples were then heat- shocked for 5 min at 37°C and pipetted into 2% Bacto-tryptone, 1% yeast extract, 0.1% NaCl, 0.02% glucose, pH 7, and incubated at 37°C with shaking for 1 h. Following centrifugation at 3200 x g for 2.5 min, the pellets were resuspended in 50 ^1 0.5% NaCl, 0.5% yeast extract, pH 7.4. The transformation products were spread onto 1.5% L-agar plates (1% NaCl, 0.5% yeast extract, 1% Bacto-tryptone, 100 fig.mV ampicillin, pH 7.4) with 50 pi of 2 % X-gal spread on top. This was incubated overnight at 37“C.
2.9.3 Colony transfer and liberation of bound DNA
Four nylon Hybond-N (Amersham) membranes were placed onto four L- agar plates (see 2.9.2 transformation) and 100 white recombinant colonies picked onto each filter and simultaneously onto a master plate. A blue colony containing non-recombinant plasmid was used as a negative control. Plates were incubated at 37°C overnight. The master plate was stored at 4*C and the filters treated to liberate the DNA from the colonies by treatment twice with 0.5 M NaOH for 2-3 min, twice with 1 M Tris-HCl (pH 7.4) for 5 min and once with 0.75 ml 1.5 M NaCl, 0.5 M Tris-HCl (pH 7.4) for 2-3 min (Grunstein and Hogness, 1975). Filters were then baked at SO’C for 2 h, and stored in 2 x SSC at 4°C.
2.9.4 Colony hybridization
The baked membranes were prehybridized for 3 h at 65“C in hybridization buffer (50 mM Na phosphate buffer, pH 6.5, 5 x SSC, 10 0.2% SDS, 250 pig.ml*’ herring sperm DNA and 5 x Denhardt’s solution containing 0.2% w/v, 0.2% Ficoll, 2% polyvinylpyrrolidone. Hybridization was at 65“C overnight in fresh buffer containing labelled probe (see Chapter 2.9.5). The hybridized membranes were washed for 1 h in 5 mM Na phosphate buffer, pH 7, 1 mM EDTA and 0.2% SDS at 50’C. The membrane was autoradiographed at -75“C between two
fragments corresponding to the 200 bp, 300 bp, 500 bp and 600 bp products (see Chapter 4) were radiolabelled with using the nick translation kit supplied by Amersham International. A reaction was set up containing 0.1 /xg target DNA, 1 nmol dCTP, 1 nmol dTTP, 1 nmol dGTP, and 1.5 U of DNA polymerase I in a total volume of 30 /xl. This was added to 20 /xCi [a-^^P]-dATP (Amersham) at 1 x
10* cpm.|xg‘ DNA specific activity, and incubated at 15®C for 2-3 h, after which the labelled DNA was separated from unincorporated radioactivity by Sephadex G50 chromatography.
The specific activity of probes was estimated by the Cerenkov method. A 1 fxl aliquot in a 1.5 ml Eppendorf tube was placed inside a glass scintillation vial and counted in a Kontron BETAmatic II liquid scintillation counter set for counting tritium. The specific activity, expressed as Ci.mmole^ DNA, assumed an
approximate efficiency of 30% counting.
2.9.6 ’Colony’ PCR
A cocktail stick was used to transfer a bacterial colony to 50 /xl of colony lysis buffer (1% Triton X-100, 20 mM Tris-HCl pH 8.5, 2 mM EDTA) and the mixture was heated to 95°C for 10 min. Following centrifugation for 2 min at 3200 X g, 2.5 fi\ of the supernatant was added to PCR reagents (2.8.3)