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From 2010.igem.org

Contents 1 CHOOOSE 2 Materials 3 Digest Mix 4 Digestion

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

Back to Protocol

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.