Colony PCR

Reaction mix (Pipett scheme)

H₂O                                         16.5 μL

10x Taq/Pfu buffer                   2 μL

Primer 1                                    0.4 μL (10 pmol)

Primer 2                                    0.4 μL (10 pmol)

dNTP-Mix                                  0.2 μL

Taq polymerase                        0.5 μL total

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                      20 μL

Thermocycler Protocol

1	Innitial melting	94 ºC	3 minutes
Start-Loop (20x Cycles)
2          Denaturation		94 ºC	20 seconds
3          Hybridization		58 ºC	20 seconds
4          Elongation		72 ºC	60 seconds per 1000 bp
Close-Loop
5	Elongation	72 ºC	5 min
6	Stop	8 ºC

Procedure

• Prepare a “Chess board” LB-plate of corresponding resistance
• For each colony:
  • lightly touch the colony with a yellow pipett-tip, on a 20µl pipett
  • Touch a numbered chess-board-tile with a pipett-tip (3 times)
  • Place the same tip in a corresponding filled PCR-reaction tube a resuspend 2-3 times
• Run PCR and incubate LB-Plate on 37°C
• Analyse PCR via Gel electrophoresis and identify positive clones
• On the same day positive clones can be expanded from the “Chess board”-LB onto a whole new plate (even if colonies are not yet visible)