How does the reaction volume determine the efficiency of restriction digestion? I have 200ng/uL insert in 30uL of buffer. What should be the reaction volume [water, buffer, enzymes (20U), DNA (30uL)] that would be optimum for restriction digestion?
Dear Tias Saha ,
Optimizing Restriction Endonuclease Reactions
There are several key factors to consider when setting up a restriction endonuclease digestion. Using the proper amounts of DNA, enzyme and buffer components in the correct reaction volume will allow you to achieve optimal digestion. By definition, 1 unit of restriction enzyme will completely digest 1 μg of substrate DNA in a 50 μl reaction in 60 minutes. This enzyme : DNA : reaction volume ratio can be used as a guide when designing reactions. However, most researchers follow the "typical" reaction conditions listed, where a 5–10 fold overdigestion is recommended to overcome variability in DNA source, quantity and purity.
To view the table of A "Typical" Restriction Digestion, please use the following link:
https://www.neb.com/tools-and-resources/usage-guidelines/optimizing-restriction-endonuclease-reactions
Enzyme
Keep on ice when not in the freezer
Should be the last component added to reaction
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.
NEB has introduced a line of High-Fidelity (HF™) enzymes that provide added flexibility to reaction setup.
DNA
Should be free of contaminants such as phenol, chloroform, alcohol, EDTA, detergents or excessive salts. Extra wash steps during purification are recommended.
Methylation of DNA can inhibit digestion with certain enzymes. For more information about methylation, visit Effect of CpG Methylation on Restriction Enzyme Cleavage and Dam and Dcm Methylases of E.coli.
Buffer
Use at a 1X concentration
Reaction Volume
A 50 µl reaction volume is recommended for digestion of 1 µg of substrate
Enzyme volume should not exceed 10% of the total reaction volume to prevent star activity due to excess glycerol
Additives in the restriction enzyme storage buffer (e.g., glycerol, salt) as well as contaminants found in the substrate solution (e.g., salt, EDTA, or alcohol) can be problematic in smaller reaction volumes. The following guidelines can be used for techniques that require smaller reaction volumes.
To view the table of Alternative Volumes for Restriction Digests, please use the following link:
https://www.neb.com/tools-and-resources/usage-guidelines/optimizing-restriction-endonuclease-reactions
Incubation Time
Incubation time is typically 1 hour
Can often be decreased by using an excess of enzyme
Can be decreased to 5-15 mins by using one of our Time-Saver Qualified enzymes.
It is possible, with many enzymes, to use fewer units and digest for up to 16 hours. For more information, visit Extended Digests with Restriction Endonucleases.
Stopping a Reaction
If no further manipulation of DNA is required:
Terminate with a stop solution (10 µl per 50 µl rxn) [2.5% Ficoll®-400, 11 mM EDTA (pH 8.0), 3.3 mM Tris-HCl, 0.017% SDS, 0.015% bromophenol blue] (i.e., NEB #B7021)
When further manipulation of DNA is required:
Heat inactivation can be used
Remove enzyme by using a spin column or phenol/chloroform extraction
Storage
Storage at -20°C is recommended for most restriction enzymes. For a few enzymes, storage at -70°C is recommended for periods longer than 30 days. Please refer to the enzyme's product page for storage information.
10X NEBuffers should also be stored at -20°C
Stability
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
Control Reactions
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.
For more information, please use the following link:
https://www.neb.com/tools-and-resources/usage-guidelines/optimizing-restriction-endonuclease-reactions
Hoping this will be helpful,
Rafik
how many units per micro liter of enzyme needed is needed to linearise the 1microliter of DNA?
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