Yeast Colony PCR

PROCEDURE

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

  2. 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

  3. 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

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