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Team:Tuebingen/Notebook/Protocols/colony-pcr
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Latest revision as of 12:05, 4 October 2013
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
- Put some PCR-tubes on ice.
- 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!
- Transfer 10 µL of master mix in every ice-cold PCR-tube (1 PCR-tube per colony).
- 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.
- Smear the tip over a new LB plate + antibiotic in order to create new colonies.
- Run PCR and control success via gelelctrophoresis.
- Store plates of successful colony-PCRs at 4 °C.
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