- 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
Notes for improving efficiency of EcoRI/SpeI Double Digest
- 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.