Restriction Enzyme Digests

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Contents

CHOOOSE

http://openwetware.org/wiki/Knight:Restriction_Digest

http://openwetware.org/wiki/Endy:Restriction_Digest

Materials

  • Restriction enzymes ([http://www.neb.com/nebecomm/products/productR0101.asp EcoR I], [http://www.neb.com/nebecomm/products/productR0133.asp Spe I], [http://www.neb.com/nebecomm/products/productR0145.asp Xba I] or [http://www.neb.com/nebecomm/products/productR0140.asp Pst I]) from [http://www.neb.com/ NEB]
  • NEB2 buffer
  • BSA
  • Nuclease free H2O

Digest Mix

Example - 50 μL reaction. 100 μL reactions are also common especially if your DNA to be cut is dilute.

  • 5 μL NEB2 buffer
  • X μL DNA ( ~500 ng).
  • 0.5 μL 100X BSA
  • 1 μL BioBricks enzyme 1
  • 1 μL BioBricks enzyme 2
  • (42.5 - X) μL deionized, sterile H2O


Digestion

  1. Digest at 37 degrees for 1.5 hours
  2. Heat kill for 20 minutes
  3. Add 1 μL Phosphotase to backbone
  4. Incubate for 30 minutes @ 37 degrees
  5. heat kill @80 degrees for 20 minutes.

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