Team:Tuebingen/Notebook/Protocols/preparative-restriction

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Preparative Restriction Digest

Reagents

10.0 µL 10x NEBuffer 2
1.0 µL 100x BSA
5 - 10 µg Plasmid DNA
2.0 µL EcoRI or XbaI
2.0 µL PstI or SpeI
to 100 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. Run gel and perform a gelextraction.

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