Colony PCR
Colony PCR
David Amberg
This procedure will work for both yeast and E. coli:
Take a small colony and suspend it in 5ul of H2O in a PCR tube. Heat for 5 min at 95¡C and then spin the condensation down in a microfuge. Set up the PCR reaction as follows:
5ul H2O + cells
5ul 5uM primer2
5ul 10XTaq Buffer
5ul 2mM dNTPs
0.5ul 10 mg/ml acetylated BSA
1ul Taq DNA polymerase
23.5 ul H2O
PCR Conditions: 94¡C x 4min. then 35 cycles of: (94¡C x 1 min then 55¡C x 1min then 72¡C x 3min) followed by 72¡C x 20 min and a 4¡C soak. (5ul run out on a mini gel should be sufficient to see product.)
10X TAQ Buffer:
0.2M TRIS pH8.3
15mM MgCl2
0.25M KCl
0.5% TWEEN 20
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