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- 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.
- 0.2-1μg 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
- Incubate reaction at 37°C for at least 1 hour; longer digest gives more complete digestion, especially if you have >1µg DNA, but can at times give nonspecific digestion, even if glycerol is <5%. Heat kill enzymes at 80C for 20 min.
- Store at -20°C.
- 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).