Restriction Digest Using A RE-Mix® Restriction Enzyme Master Mix
|RE-Mix Master Mix
|Total Reaction Volume
- Keep on ice when not in the freezer
- Mix components by pipetting the reaction mixture up and down, or by "flicking" the reaction tube. Follow with a quick ("touch") spin-down in a microcentrifuge. Do not vortex the reaction.
- In general, we recommend 5–10 units of enzyme per µg DNA, and 10–20 units for genomic DNA in a 1 hour digest.
- RE-Mix Master Mixes should be used at 1X concentration.
- RE-Mix Master Mixes already include buffer and BSA so no additional buffer is supplied.
- A 20 µl reaction volume is recommended for digestion with a RE-Mix Master Mix.
- The recommended incubation time with a RE-Mix Master Mix is 15 minutes.
Stopping a Reaction
- The RE-Mix Master Mix includes a density agent and dye and does not require addition of a stop solution.
When further manipulation of DNA is required:
- Heat inactivation can be used.
- Remove enzyme by using a spin column or phenol/chloroform extraction.
- RE-Mix Master Mixes should be stored at -20°C.
- All enzymes are assayed for activity every 4 months. The expiration date is found on the label.
- Exposure to temperatures above -20°C should be minimized whenever possible.
If you are having difficulty cleaving your DNA substrate, we recommend the following control reactions:
- Control DNA (DNA with multiple known sites for the enzyme, e.g. lambda or adenovirus-2 DNA) with restriction enzyme to test enzyme viability
- If the control DNA is cleaved and the experimental DNA resists cleavage, the two DNAs can be mixed to determine if an inhibitor is present in the experimental sample. If an inhibitor (often salt, EDTA or phenol) is present, the control DNA will not cut after mixing.
Re-Mix® is a registered trademark of New England Biolabs, Inc.