Team:Amsterdam/Notebook/Protocols/ColonyPCR

From 2011.igem.org

Colony PCR

We used this protocol to check if the new transformants contained the right plasmids/insert.

Materials

  • Petri dish with LB agar and appropriate antibiotic
  • ON colonies on a petri dish with LB agar and appropriate antibiotic
  • dNTPs
  • primers (VF2 and VR)
  • Taq polymerase
  • Taq buffer
  • dH2O
  • PCR tubes
  • PCR machine

Protocol

Sample µl
VF2 primer 0,5
VR primer 0,5
dNTPs 1
Taq buffer 2,5
Taq polymerase 0,2
H2O 20,3
Total 25 µl

  1. Make the PCR mix and pippete it in a PCR tube.
  2. Pick a colony from a LB-agar plate using a sterile pipette tip.
  3. Pippete up and down in the PCR mix.
  4. Cross the pippete tip on the new LB-agar plate in the box corresponding to the sample number.
  5. Redo this with all the selected colonies.
  6. Run the following PCR program:

Colony PCR.jpg
Note: Adjust the elongation time according to the expected product size. 1 minute for each Kb.

  1. Incubate the selected colonoies on the LB-agar plate ON at 37°C.
  2. Check the PCR-sample sizes on agarose gel.





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