General scheme for PCR-reactions

Always set up a negative control (no template DNA)

Always use a fresh tip for each step

Unless using HotStar Taq, set up reactions at 4°C

H20

10x PCR-buffer 10µl each

dNTPs (dATP, dTTP, dGTP, dCTP) (25 mM) 0.8 µl each

Primers 100 ng/ul 2.5 µl each

Template 100ng/ul 1 µl

Taq/Pfu polymerase 1Unit (U)

Volume 100 µl

PCR-conditions: see booklet

in general: 1 min @ 95°C (denaturation)

1 min @ (Tm-5°C) (annealing)

1-2 min/kb @ 72°C (extension)

25 cycles

then 72°C for 10 min