Endy:Restriction Digest

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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])
*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])
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*Restriction buffers  
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*Restriction buffers (also from New England Biolabs. [http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/buffer_activity_restriction_enzymes.asp? source])
**EX – EcoR I & BSA
**EX – EcoR I & BSA
**ES – EcoR I  
**ES – EcoR I  
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**SP – 2 & BSA
**SP – 2 & BSA
**ddH<sub>2</sub>O
**ddH<sub>2</sub>O
 +
''Notes for improving efficiency of [[EcoRI/SpeI Double Digest]]''
''Notes for improving efficiency of [[EcoRI/SpeI Double Digest]]''

Revision as of 11:48, 20 June 2006

Materials

  • Need roughly 3-5 μL of prepped DNA. Aim for 1 μg of DNA.
  • Restriction buffers (also from New England Biolabs. source)
    • EX – EcoR I & BSA
    • ES – EcoR I
    • XP – 3 & BSA
    • SP – 2 & BSA
    • ddH2O


Notes for improving efficiency of EcoRI/SpeI Double Digest

Digest Mix

  • μg DNA from prep or PCR
  • 10% (by volume) Restriction Buffer
  • 1% (by volume) BSA stock
  • 2.5% - 5% (by volume) of each restriction enzyme.
  • ddH2O to produce correct percentages by volume

Enzymes < 10% of total volume so glycerol is < 5% of total volume. At higher glycerol concentrations, star activity is sometimes observed (meaning that the enzyme cuts at places other than its recognition site).

Example - 20μl mix

  • 2.0 μL Restriction Buffer
  • 0.2 μL BSA stock
  • 3-5 μL DNA from prep or PCR
  • 0.5 μL Restriction Enzyme Stock
  • X μL ddH2O (to bring solution up 20μL)

Incubate reaction at 37°C overnight (minimum 4-6 hours).

Store at -20°C.

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