Endy:Restriction Digest

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==Materials==
==Materials==
-
*Need roughly 3-5 μL of prepped DNA. Aim for 1 μg of DNA.
+
*Prepped DNA. For a BioBrick assembly (and possibly any cloning operation) 300 ng is more than enough.
*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])
*Restriction buffers (also from New England Biolabs. [http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/buffer_activity_restriction_enzymes.asp? source])
*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
+
**For all combinations of the four BioBrick enzymes, we use Buffer 2 + BSA.
-
**ES – EcoR I
+
*ddH<sub>2</sub>O
-
**XP – 3 & BSA
+
-
**SP – 2 & BSA
+
-
**ddH<sub>2</sub>O
+
-
 
+
-
 
+
-
''Notes for improving efficiency of [[EcoRI/SpeI Double Digest]]''
+
==Digest Mix==
==Digest Mix==
-
 
+
*ng DNA from prep or PCR
-
* &mu;g DNA from prep or PCR
+
* 10% (by volume) Restriction Buffer
* 10% (by volume) Restriction Buffer
* 1% (by volume) BSA stock
* 1% (by volume) BSA stock
* 2.5% - 5% (by volume) of each restriction enzyme.
* 2.5% - 5% (by volume) of each restriction enzyme.
* ddH<sub>2</sub>O to produce correct percentages by volume
* ddH<sub>2</sub>O to produce correct percentages by volume
 +
*Enzymes < 10% of total volume so glycerol is < 5% of total 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).''
+
We prepare a restriction digest supermix (stored at -20C) containing -
 +
*5*'''n''' &mu;l of Buffer 2
 +
*0.5*'''n''' &mu;l of BSA
 +
*37*'''n''' &mu;l of ddH<sub>2</sub>O
 +
where '''n''' is the desired number of 50&mu;l reactions.
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'''Example - 20&mu;l mix'''
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===Example - 50&mu;l digest===
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*2.0 &mu;L Restriction Buffer
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*42.5&mu;l of restriction digest supermix
-
*0.2 &mu;L BSA stock
+
*1-5&mu;l of preppedDNA
-
*3-5 &mu;L DNA from prep or PCR
+
*1&mu;l of each restriction enzyme
-
*0.5 &mu;L Restriction Enzyme Stock
+
-
*X &mu;L ddH<sub>2</sub>O (to bring solution up 20&mu;L)
+
-
Incubate reaction at 37&deg;C overnight (minimum 4-6 hours).
+
==Reaction conditions==
 +
*Incubate reaction at 37&deg;C for 1 hour.  Heat kill enzymes at 80C for 20 min.
 +
*Store at -20&deg;C.
-
Store at -20&deg;C.
+
==Notes==
 +
*On improving the efficiency of [[EcoRI/SpeI Double Digest]].
 +
*At glycerol concentrations > 5%, star activity is sometimes observed (meaning that the enzyme cuts at places other than its recognition site).

Revision as of 12:11, 20 September 2007

Contents

Materials

  • Prepped DNA. For a BioBrick assembly (and possibly any cloning operation) 300 ng is more than enough.
  • Restriction buffers (also from New England Biolabs. source)
    • For all combinations of the four BioBrick enzymes, we use Buffer 2 + BSA.
  • ddH2O

Digest Mix

  • ng 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.

We prepare a restriction digest supermix (stored at -20C) containing -

  • 5*n μl of Buffer 2
  • 0.5*n μl of BSA
  • 37*n μl of ddH2O

where n is the desired number of 50μl reactions.

Example - 50μl digest

  • 42.5μl of restriction digest supermix
  • 1-5μl of preppedDNA
  • 1μl of each restriction enzyme

Reaction conditions

  • Incubate reaction at 37°C for 1 hour. Heat kill enzymes at 80C for 20 min.
  • Store at -20°C.

Notes

  • On improving the efficiency of EcoRI/SpeI Double Digest.
  • At glycerol concentrations > 5%, star activity is sometimes observed (meaning that the enzyme cuts at places other than its recognition site).
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