Infecciones virales agudas de glándulas salivales

4.1.1 Spectrometric Determination of DNA Concentration

Double-stranded DNA has an absorption maximum at 260 nm; by measuring the absorption of an aequous DNA dilution in a 1 cm quartz cuvette, the DNA concentration of the sample could be calculated using the following formula:

conc. DNA [μg/ml] = OD260 * 50 * dilution factor

4.1.2 Gene Amplification by PCR

Primer annealing temperatures varied in different reactions and were chosen according to the predicted primer melting temperature which is influenced by the GC content of the primers' overlapping regions.

PCR reaction mix:

50-60 ng plasmid DNA as reaction template 1 μl dNTP solution (12.5 mM)

2 μl primer No. 1 (50 μM) 2 μl primer No. 2 (50 μM)

5 μl polymerase buffer (10x, provided by manufacturer) 38 μl H2O

1 μl (2 U) Vent polymerase.

Reaction cycles: 5 min heating to 95°C; 30 amplification cycles:

30 sec 95°C: melting of double-stranded DNA

30 sec annealing of primers; temperatures varying from 52-62°C 2 min 72°C: DNA synthesis

MATERIALS AND METHODS 24

4.1.3 Site-directed Mutagenesis by PCR

In order to achieve a single amino acid exchange in a protein, oligonucleotide primers were designed that introduced a point mutation in the desired DNA codon. A mutagenic primer pair contains the same DNA sequence in opposite directions, while the mutated base is located in the middle with about 10-15 additional bases of the original DNA sequence on both sides. The primer length was chosen so as to reach a melting temperature of about 78°C after the following formula: Tm= (2°C)(A+T) + (4°C)(G+C). Care was taken that the primers' GC content amounted to at least 40% and that both primer ends contained GC bases when possible. In the PCR reaction, primer extension times were set to 2 minutes per 1000 base pairs of template DNA.

PCR reaction mix:

5-50 ng plasmid-DNA as reaction template 0.8 μl dNTP solution (12.5 mM)

2.5 μl primer No. 1 (50 ng/μl) 2.5 μl primer No. 2 (50 ng/μl)

5 μl Pfu polymerase buffer (10x, provided by manufacturer) 37.5 μl H2O

1 μl (2.5 U) Pfu polymerase.

Reaction cycles: 30 sec heating to 95°C; 16 amplification cycles:

30 sec 95°C: melting of double-stranded DNA 1 min 55°C: annealing of primers

2 min per 1kb template at 68°C: DNA extension Reaction termination by cooling to 15°C.

In order to get rid of unmutated template DNA, 20 μl of the reaction mix were incubated at 37°C for 2 hours with 1 μl (20 U) Dpn1 restriction enzyme which cuts the unwanted methylated plasmid DNA. 2 μl of this restriction assay were used for transformation in bacteria.

MATERIALS AND METHODS 25

4.1.4 Restriction of DNA

DNA restriction was used to generate vector or insert fragments necessary for DNA cloning. Insert and vector were cut with either one or two restriction enzymes at a time. 0.5 - 1 μg DNA for analytical purposes or about 10 μg DNA for larger preparations were cut with 1 - 5 U restriction enzyme per μg DNA. Buffers were used according to the manufacturer's protocols; to avoid unspecific cutting, the volume of each reaction was raised to a minimum of 10x the amount of restriction enzymes used. Incubation times were at least 3 hours, larger amounts of DNA overnight at the temperature suitable for the respective enzymes. The efficiency of the restriction was controlled by running a test sample on an agarose gel.

4.1.5 Ligation of DNA Fragments

In DNA ligations, the ratio of insert to vector should be at least 3:1; DNA concentrations were determined by agarose gel electrophoresis. The ligation reaction mix was incubated overnight at 16°C; about 1.5 μl of the solution were later used for transformation in bacteria.

Ligation assay: 0.5 μl vector DNA 3 μl insert DNA

1.5 μl ligase buffer (10x; provided by manufacturer) 9 μl H2O

1 μl (400 U) T4 DNA Ligase

4.1.6 Preparation of Competent E. Coli

4 ml LB medium were inoculated with the desired strain of E. coli and grown at

37°C overnight. The following morning, the culture was transferred into 300 ml LB medium and grown to a OD600 of 0.55, the flask then placed in ice for 20 minutes, and the

cells harvested by centrifugation at 2500 g and 4°C for 20 minutes. The medium was completely poured off and remaining drops of medium were removed by putting the open bottles on paper towels for a short time. The cells were resuspended in about 60-80 ml of refrigerated Inoue transformation buffer and harvested by centrifugation at 2500 g and 4°C for 15 minutes. The medium was once again poured off, the cell pellet resuspended in about 20 ml of refrigerated Inoue transformation buffer, and 1.5 ml DMSO added to the

MATERIALS AND METHODS 26

suspension. After 10 minutes incubation on ice, the cells were transferred in small aliquots of 50 μl into pre-cooled tubes, frozen in liquid nitrogen, and stored at -80°C until usage.

4.1.7 Quick Transformation of Chemically Competent E. Coli

Chemically competent E. coli cells kept in 50 μl aliquots at -80°C were thawed on

ice, mixed with a small amount of the desired DNA plasmid (1-3 μl), and incubated on ice for about 20 minutes. A heat shock was applied to the cells by transferring them into a 42°C water bath for 2 minutes, and afterwards they were again incubated on ice for about 10 minutes. The cells were then diluted in about 150 μl of water or LB medium, and immediately plated onto LB agarose plates containing the appropiate antibiotic substance for selection.