Team:Tuebingen/Notebook/Protocols/ligation

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Protocols

DNA-Ligation

Reagents

20-50 ng linearized vector
1 µL 10x ligation buffer
1 µL T4 ligase
1 µL T4 ligase
1 µL ATP
100-200 ng Insert
to 10 µL H2O

 

Procedure

  1. Mix all reagents in a PCR-tube and if necessary add to 10 μl with H2O.
  2. Run cycler starting at 25 °C, reducing temperature hourly by 1 degree and remaining at 4 °C.
  3. Heat inactivate the enzymes for 5 min at 70 °C.
  4. Store at -20 °C.

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