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