Restriction Enzyme Digests

From 2010.igem.org

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http://openwetware.org/wiki/Endy:Restriction_Digest
http://openwetware.org/wiki/Endy:Restriction_Digest
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After heat-killing restriction enzymes, add dpni to rid of background plasmids (circular template plasmids that are supposed to be linearized) - dpni works in most buffers
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==Materials==
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*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]
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*NEB2 buffer
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*BSA
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*Nuclease free H<sub>2</sub>O
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==Digest Mix==
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''Example - 50 &mu;L reaction.  100 &mu;L reactions are also common especially if your DNA to be cut is dilute.''
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*5 &mu;L NEB2 buffer
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*X &mu;L DNA ( ~500 ng).
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*0.5 &mu;L 100X BSA
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*1 &mu;L BioBricks enzyme 1
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*1 &mu;L BioBricks enzyme 2
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*(42.5 - X) &mu;L deionized, sterile H<sub>2</sub>O
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==Digestion==
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# Digest at 37 degrees for 1.5 hours
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# Heat kill for 20 minutes
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# Add 1 &mu;L Phosphotase to backbone
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# Incubate for 30 minutes @ 37 degrees
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# heat kill @80 degrees for 20 minutes.
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Back to [[Team:Northwestern/Protocol|<span style="color:#4B088A;">Protocol</span>]]
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[[Category:Protocol]]

Latest revision as of 21:05, 16 October 2010

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.

Back to Protocol

Retrieved from "http://2010.igem.org/Restriction_Enzyme_Digests"