Restriction digestion¶
NEB¶
Overview¶
- rCutSmart buffer (pH 7.9 @ 25C):
- 50 mM K-acetate
- 20 mM Tris-acetate
- 10 mM Mg-acetate
- 100 µg/ml albumin
Procedure¶
- Add and flick the tube:
| Reagent | Amount |
|---|---|
| Template DNA | 1 pmol |
| 10X NEBuffer / rCutSmart Buffer | 5 uL |
| Restriction enzyme | 1 uL |
| Milli-Q | to 50 uL |
- 1 ug of template DNA = 100 ng of short DNAs (~100 bp) = 1 pmol.
- Empirically, even 3 pmol are successfully digested when left for 8 hours.
- 1 unit of restriction enzyme will completely digest 1 μg of substrate DNA in a 50 μl reaction in 60 minutes
- 5-10 units of enzyme recommended per 1 ug of DNA
-
Enzyme volume should not exceed 10% of the total reaction volume to prevent star activity
-
Incubate at 37C (enzyme-dependent) for 1 hour (enzyme-dependent)*.
- For Time-Saver Qualified enzymes, can incubate for 5-15 min, but can also leave overnight.
- Empirically, smaller fragments will be successfully digested if left for 8 h.
- Heat-inactivate at 65-80C for 20 min (temperature is enzyme-dependent).
Resources¶
- BsaI-HF®v2
- Optimizing Restriction Endonuclease Reactions
- Cleavage Close to the End of DNA Fragments
- Activity of Restriction Enzymes in PCR Buffers
- NEBuffer Activity/Performance Chart with Restriction Enzymes (+ incubation and inactivation temperatures)
ThermoFisher¶
BfuI / BciVI 1. Add:
| Reagent | Amount (uL) | Amount if using PCR products directly (uL) |
|---|---|---|
| Nuclease-free water | 16 | 18 |
| 10X Buffer BfuI | 2 | 2 |
| DNA | 1 (0.5-1 μg/μL) | 10 (~0.1-0.5 μg of DNA) |
| BfuI | 0.5-2 | 1-2 |
- Mix gently and spin down for a few seconds.
- Incubate at 37°C for 1-16 hours.
- An excess of BfuI (7.5 U/μg DNA x 1 hour) may result in star activity.
- 1U is defined as the amount of BfuI required to digest 1 μg of lambda DNA in 1 hour at 37°C in 50 μL of recommended reaction buffer.
- Bfu concentration is 5 U/uL