Team:Tuebingen/Notebook/Protocols/3A assembly

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Three part assembly

Digestion

Reagents

500 ng part plasmid DNA
0.5 µL of each restriction enzyme
2.5 µL 10x NEB buffer 2
0.25 µL 100x BSA
to 25 µL H20

Procedure

  1. After mixing the reagents in a 1,5 ml Eppendorf-tube: Incubation at 37 °C overnight.
  2. Heat inactivate the restriction enzymes at 80 °C for 20 min.

 

Ligation

Reagents

1 µL 10x ligation buffer
100-200 ng upstream part plasmid
100-200 ng downstream part plasmid
50 ng destination part plasmid
1 µL T4 ligase
1 µL ATP
to 10 µL H20

Procedure

  1. Mix all reagents in a PCR-tube and if necessary add to 10 μl with H20.
  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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