Endy:Restriction Digest: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
No edit summary |
No edit summary |
||
| Line 5: | Line 5: | ||
*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 | *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 | ||
| Line 11: | Line 11: | ||
**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 09:48, 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.
