Colony PCR

Method 1

  1. Prepare PCR mixture:

    Taq Polymerase Reactions (50 ul):
    • 32.2 ul distilled H2O
    • 3.4 ul 1M Tris, pH8.5
    • 0.8 ul 1M NH4SO4
    • 0.5 ul 1% Triton X-100
    • 8.3 ul 30% glycerol
    • 1.25 ul Mg(Ac)2
    • 1 ul 10mM dNTPs
    • 100 pmoles for primer1, usually 1 ul of a 100pM stock
    • 100 pmoles for primer2, usually 1 ul of a 100pM stock

    Note: Volume of distilled H2O can be adjusted if additional volumes of reagents are required.

  2. Before adding Taq Polymerase, pick a healthy chunk of a colony using a pipet tip and mix with the appropriate PCR mix.

  3. Add 0.5 ul Taq DNA Polymerase to each reaction.

  4. PCR cycles as follows:

    94oC for 3 minutes
    94oC for 30 seconds
    55oC for 30 seconds
    72oC for 45 seconds
      ==> 25-35 cycles
    72oC for 7 minutes
    4oC hold

    Note: The incubation times at each temperature can be varied to accommodate different volumes of reactions.
    The elongation times (at 72oC) work well for amplification of loci <1.5 kbp in size. These conditions may need to be modified for amplification of longer regions.

    Also, annealing temperature may have to be determined emperically.

  5. Check PCR on a gel.

Method 2

  1. Prepare eppendorf tubes containing 20 ul 0.25% SDS.

  2. Use pipette tip to transfer yeast cells (about 1E7) into the tubes. Vortex briefly and centrifuge for 30 seconds. (Optional) You can heat treat the sample at 90oC for 3 minutes if you choose.

  3. Prepare PCR mixture:

    Taq Polymerase Reactions (50 ul):
    • 39.42 ul distilled H2O
    • 0.63 ul 1M Tris, pH8.5
    • 0.95 ul 3M KCl
    • 1 ul 10mM dNTPs
    • 2 ul 100mM MgCl2
    • 2.5 ul 20% Triton X-100
    • 100 pmoles primer1, usually 1 ul of a 100pM stock
    • 100 pmoles primer2, usually 1 ul of a 100pM stock
    • 1 ul genomic extract
    • 0.25 ul Taq Polymerase (optional)

    Note: Volume of distilled H2O can be adjusted if additional volumes of reagents are required.

  4. PCR Cycles (for PE 9600 or equivalent):
    94oC for 3 minutes
    94oC for 30 seconds
    55oC for 30 seconds
    72oC for 45 seconds
      ==> 25-35 cycles
    72oC for 7 minutes
    4oC hold

    Note: The incubation times at each temperature can be varied to accommodate different volumes of reactions.
    The elongation times (at 72oC) work well for amplification of loci <1.5 kbp in size. These conditions may need to be modified for amplification of longer regions.

    Also, annealing temperature may have to be determined emperically.

  5. Check PCR on a gel.