School of Engineering and Science - Jacobs University

Material and Methods
stained in ethidium bromide solution (10 µg/ml in TAE buffer) for 5-15 min.
Then the gel was analysed and documented under UV-light using the gel jet
imager system (INTAS).
4.2.4 Polymerase chain reaction (PCR)
Polymerase chain reaction (PCR) is a method used to amplify a specific DNA
sequence in vitro by repeated cycles of synthesis with specific primers and
thermostable Taq DNA polymerase (Saiki et al., 1988). Specific primers are
complementary to sequences that lie on opposite strands of the template DNA
and flank the segment 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 primers and dNTPs. The reaction mixture was then cooled to a
temperature that allows the primers to anneal to their target sequences, and
subsequently the annealed primers were extended with DNA polymerase.
Cycles of denaturation, annealing, and DNA synthesis were repeated several
times. Reaction mixture was prepared according to the supplier’s manual.
As the primers used in this study were designed from heterologues sequence
information, touch-down PCR was applied to enhance the yield of product.
A typical PCR thermoprofile was programmed as follows:
Initial denaturation 95°C 5 min 1x
Denaturation 95°C 1 min
Hybridisation 65-50° 30 sec. 10x
Polymerisation 65°C 1 min/kb
Denaturation 95°C 1 min
Hybridisation 55°C-45°C (-0.5°C/cycle) 30 sec. 25x
Polymerisation 65°C 1 min/kb
Final extension 65°C 5 min
32