Team:Tuebingen/Notebook/Protocols/colony-pcr

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Colony-PCR

Reagents

0.5 µL MgCl2 (c = 25 mM)
1.0 µL 10x Taq Polymerase Buffer
0.1 µL dNTPs (2.5 mM each)
0.1 µL Oligo forward
0.1 µL Oligo reverse
7.7 µL Aqua dest.
0.1 µL Taq Polymerase (5 U/µL)

 

Procedure

  1. Put some PCR-tubes on ice.
  2. The given volumes are for one single reaction of 15 µL. When checking more than one colony, repare a master mix for all colonies. Keep that master mix on ice all the time!
  3. Transfer 10 µL of master mix in every ice-cold PCR-tube (1 PCR-tube per colony).
  4. Usa a pipet tip in order to pick up a colony from a fresh plate and transfer the colony into the reaction mixture inside a PCR-tube.
  5. Smear the tip over a new LB plate + antibiotic in order to create new colonies.
  6. Run PCR and control success via gelelctrophoresis.
  7. Store plates of successful colony-PCRs at 4 °C.

 

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