Endy:Restriction Digest: Difference between revisions
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==Materials== | ==Materials== | ||
* | *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]) | ||
** | **For all combinations of the four BioBrick enzymes, we use Buffer 2 + BSA. | ||
*ddH<sub>2</sub>O | |||
==Digest Mix== | ==Digest Mix== | ||
*ng 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. | |||
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 ddH<sub>2</sub>O | |||
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 | |||
* | |||
Incubate reaction at 37°C | ==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). | |||
Revision as of 09:11, 20 September 2007
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).
