PROCEDURE

1. Add 1 µl Zymolyase 100T 10 mg/ml to 9 µl H2O and suspend a yeast colony in this solution.

2. Add 40 µl PCR reaction mix to the suspended colony:

   5 µl 5 mM gene specific primer 1
   5 µl 5 mM gene specific primer 2
   5 µl 10 x Taq buffer
   5 µl 2 mM dNTPs
   0.5 µl 10 mg/ml BSA
   1 µl Taq
   18.5 µl H2O

3. PCR cycle profile:

   94°C 4 min
   ---------------------------------------------------------
   94°C 1 min
   55°C 1 min
   72°C 1 min per kb
   >>>35 cycles
   ---------------------------------------------------------
   72°C for 10 min

4. Load 10 µl or more on a gel.

MATERIALS AND SOLUTIONS

10 X Taq Buffer:

0.2 M TRIS pH 8.3
15 mM MgCl2
0.25 M KCl
0.5% Tween 20