Colony PCR


As an alternative to minipreps you may test the clones of a ligation/transformation procedure by PCR. Bacteria from a single clone are suspended in a PCR mix with a primer pair that identifies correct constructs. Withoug special lysis of the bacteria the PCR reaction is performed and analysed by agarose gel electrophoresis.

  1. Prepare the PCR reaction mix with 12.5 µl per reaction and about 50 sample.
    2. 10 µl water
    0.5 µl 10 µM primer stock (final 0.4 µM each)
    0.25 µl 10 mM dNTP mix (final each 0.2 mM)
    1.25 µl 10x Taq buffer
    0.5 µl Taq polymerase
  2. distribute the reaction mix into PCR vials
  3. with a tooth pick, pick bacteria from a single colony
  4. suspend bacteria in the PCR vial, then streak out on a appropriatly labelled LB plate
  5. run the PCR with 35 cycles (add 2 min 94°C prior to the actual cycle)
  6. analyse by agarose electrophoresis
  7. incubate the LB plate overnight at 37°C