Team:Tuebingen/Notebook/Protocols/control-restriction

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Control Restriction Digest

Reagents

1.5 µL 10x NEBuffer 2
0.15 µL 100x BSA
3 µL DNA from Mini-Prep (c > 50 ng/µL)
0.15 µL EcoRI or XbaI
0.15 µL PstI or SpeI
to 15 µL Aqua dest.

 

Procedure

  1. Around noon, mix all reagents in an 1.5 mL Eppendorf-tube and incubate at 37 °C until afternoon.
  2. 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 control-gel in order to check a ligation / transformation.

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