PCR was performed in 50 
l volumes using 0.5 g of genomic DNA
extracted from whole blood (see section DNAprep). Added were 4 l of a dNTP
mix (200 M each, Pharmacia), 5 l tenfold Taq-polymerase buffer
(Promega), 1.5mM MgCl 
(3 l 25mM MgCl ) and 2.5 units Taq-DNA
polymerase (Promega). The
polymerase buffer consisted of 50mM KCl, 10mM Tris 
HCl (pH 9.0 at room
temperature) and 1% Triton X-100 . Both primers ( 
25 and
231) were used at a final concentration of 0.5 M each (25 pmol
added).
The reaction
volume was mixed, shortly centrifuged and overlayed with 30 l mineral oil.
After amplification, the mineral oil was removed by extraction with 100 l
chloroform and the amplified DNA precipitated with two volumes absolute ethanol
(see section EtOH). The isolated pellet was then resuspended in 15
l water and a 4 l sample was visualized on an agarose-gel to
ensure positive reaction together with a negative control (PCR mixture without
added template).
The primers had an integrated EcoRI linker for subcloning:
The following table summarizes the characteristics of the used primer pair
25/ 231. The theoretical melting point
T 
can be
calculated using different methods, all three referenced in section
primertemp
are given.
Table 3.2: Cloning primers characteristics
For cycle protocol, an initial denaturation step (95psy176 C for 2 minutes) was followed by an annealing step (53psy176 C), extension step (72psy176 C) and denaturing step (94psy176 C), each of one minute. After 35 cycles, a final extension step (72psy176 C) of 5 minutes was added.