Team:Tuebingen/Notebook/Protocols/3a-assembly

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3A-Assembly
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Digestion

Reagents

2.5 µL 10x NEBuffer 2
0.25 100x BSA
250 - 300 ng Plasmid DNA
0.5 µL EcoRI or XbaI
0.5 µL PstI or SpeI
to 25 µL Aqua dest.
1/10 vol 10X Antarctic Phosphatase

 

Procedure

  1. Mix all reagents (except for Antarctic Phosphatase) in an 1.5 mL Eppendorf-tube and incubate at 37 °C over night.
  2. On the next day, heat inactivate restriction enzymes at 80°C for 20 min.
  3. Add 1/10 volume of 10X Antarctic Phosphatase and mix. Incubate at 37 °C for 15 min for 5' extensions.
  4. Heat inactivate phosphatase at 70 °C for 5 min.
  5. Perform the previous steps for upstream part plasmid, downstream part plasmid, and destination part plasmid. Digest upstream part with EcoRI / SpeI, downstream part with XbaI / PstI, and destination part plasmid with EcoRI / PstI.

 

Digestion


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