The size and the concentration of the horizontal slab gels was chosen with regard to the nature of the sample needed to be analysed. Usually 0.8% or 2% agarose gels were used. In general mini-gels (40 ml) were used for rapid electrophoresis, and for examining the PCR products (on 2% gel) unless the band of interest needed to be cut from the gel in order to be cleaned and used for sequencing, it was ran again on 0.8% gel. Larger gels (200 ml) were used for a more accurate resolution. The agarose was dissolved in
Ethidium bromide was added to the media at a final concentration of 0.5 piglmX. The gel was poured into the appropriate gel holder, and the required comb providing the sample wells was placed in its position. The gel was allowed to solidify at room temperature. After solidification of the gel, the comb was removed and the gel was submerged in 1 X TAE buffer. The DNA samples were loaded to the gel wells by using the micropipette, and the electrophoresis was performed at 25 volts (mini gel) for 2 h or 75 volts (midi gel) for 3 to 4 h, depending on the purpose of running the gel (high resolution was achieved at lower voltages whilst fast separation was achieved at higher voltages.
E lectrop horesis B uffer,
10 X TAE (Tris-acetate-EDTA). 48.4 gm of Tris-base, 11.42 ml glacial acetic acid, and 20 ml of 0.5 M EDTA. The total vol was continued to 1 litre, and the pH was adjusted to 8.0.
L oading B uffer.
0.25% brom ophenol blue, 0.25% xylene cyanol, 30% glycerol in water, and the buffer was stored at 4°C.
C racking B uffer.
2 ml of 5 M NaOH, 2.5 ml 10% SDS, 10 gm sucrose, Bromocrysol green as a dye, and the total vol was made up with distilled water to 50 ml.
L ysis B uffer.
50 mM glucose, 25 mM Tric-HCl pH 8.0, and 10 mM
EDTA.
2.4.2 Polym erase Chain Reaction (PCR) Principle.
PCR was used to amplify DNA fragment that lies between two regions of known sequence. In every PCR reaction two oligonucleotides were used as primers for a series of synthetic reactions, that are catalysed by a DNA polymerase. These primers have different sequences (see section 2.4.2.1), and were complementry to sequences that lie on opposite strands of the template DNA and flank the fragment of DNA that is to be amplified. The template DNA was first denatured by heating in the presence of a large molar excess of each of the two oligonucleotides and the
4 dNTPs. The reaction mixture was cooled to a temperature which allows the primers to anneal to their target sequences. After that the annealed primers were elongated with DNA polymerase. The cycle of dénaturation, annealing, and DNA synthesis was repeated several times (see section PCR conditions). Because the product of one cycle of amplification serves as templates for the next, each successive cycle doubles the amount of DNA product. The presence of bacterial sequences in putative fungal transformants was confirmed by the ability to amplify such sequences using the PCR method. Different primers (Ampicillin, or lambda (k) ) were used depending on the clone used to transform the fungal strain. The enzyme used was Taq polymerase. For DNA sequencing PCR was used to amplify
fragments of the cnxH gene from wild-type genomic DNA. Additionally, DNA fragments of temperature-conditional or non conditional cnxH mutants were amplified for the same purpose. In every single PCR reaction, wild-type (as a positive control) and double autoclaved distilled water (as a negative control) were included. The products of PCR reactions were providing template for cloning, and direct sequencing of PCR amplified regions of the gene. The universal PCR primers used for sequencing wild-type
cnxH gene were provided by Amersham. Other primers were designed from the sequence of the wild-type gene, and provided by
either Amersham or St. Andrews university.
The PCR M ethod.
PCR reactions were carried out in a total vol of 100 /d, in double autoclaved PCR Eppendorf tube. In order to ensure that a maximal yield of product with the correct size of band is achieved, a series of experiments were carried out to determine the optimum quantity of DNA template (ranging from 1 pg to 1 pig) and the volume of concentrated primer (0.5-1 pil). The reaction conditions for each set of primers were also optimised individually with respect to annealing temperatures, extension times, and cycle number. The PCR reactions were carried out in a HYBAID thermal cycler.
2 .4 .2 .1 Preparation O f Prim ers.
a ) DNA pellet was resuspended in 10 pi\ sterile distilled water and
incubated for 15 min, at room temperature.
b) Primer concentration was estimated spectrophotometrically at 260 nm, and was calculated according to the following equation: OD260 X Dilution Factor
--- ]Sj. Primer Base Number. 0.01 X N
c) The primer was diluted suitably to a working concentration of 1 pmol/jwl, and used for PCR amplification.
P rim ers U sed F or A m p lific a tio n O f
A nidulans
T ransform ants.Primer Used Primer sequence Annealing Temperature
AMPl CFG TGA CFG GTG AGT AC 52° AMP2 CAA CAT TTA CGT GTC GC 52° XI GTT CTC AAT TTC AGC ATC C 54° X2 GAC AGG TGC TGA AAG CG 54° AMPl-AMP2 2165 - 2477 312 bp (Acc.no.x02514) XI - X2 120 - 367 247 bp (Acc.no. voo636)
AMP1+ AMP2 carried on pUC, pBluescript, pWE15 vectors. XI + X2 carried on pWE15 vector.
The primer is up to 20 - 22 mer (units). Annealing temperature depends on the number of G and C in the primer, i.e 2(A+T) + 4(G+C)= annealing temperature.
Prim ers Used For DNA Sequencing.
Prim ers used for sequencing
cnxH.
F CCA TCC TGT CAA GTC TTG. F2 AAT GAC TGG AAG AGA GA F3 ACC CAT CCC TCT CCC TG F4 GCG ACG GGA CGG TCA TC F5 TGC GTT GCG TTC GGA TC F6 AAT AAC TGA CFG AAC TG F7 GTA ACC AGC GTA CAT TG F8 CGC CTC TTG AAC TCC AG
R GGC AGT GCA GTT CAA GC