Quality Guarantee:
HELINI Restriction endonucleases are of consistent quality as they pass the standard quality control assays (Non-specific nuclease and Cross-contamination assay, Blue/White Cloning assay) and in addition the Labelled Oligonucleotide) test to guarantee that they are free from trace amounts of endo & exo-deoxyribonucleases and phosphatase.
Activity Assay:
One unit of Restriction enzyme is the amount required to hydrolyze 1µg of DNA in 60 minutes in a total reaction volume of 50µl. Concentrated enzymes are first diluted to approximately 500-1000units/ml with enzyme dilution buffer before determination of activity. Reactions have been optimized at 37ºC using lambda phage DNA as a substrate.
Storage and Shipping:
All restriction endonucleases must be stored at -20ºC. During shipment in dry ice, enzyme may freeze. This does not affect their quality, since all our enzymes retain their activity after three freeze thaw cycles. Short time exposure to +4ºC does not affect enzymes quality.
Concentration:
Most of restriction enzymes are supplied at a user friendly concentration of 10u/µl.
A typical Restriction Digest
Most researchers follow the general rule that 10units of restriction endonucleases is sufficient to overcome variability in DNA source, quantity and purity. Generally, 1µl of enzyme is added to 1µg of purified DNA in a final volume of 50µl of the appropriate 1X Buffer followed by incubation for 1 hour at the recommended temperature. If an excess of enzyme is used, the length of incubation can often be decreased to save time. Alternatively you can productively digest with fewer units of enzyme for up to 16 hours with many restriction enzymes.
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Setting up a Restriction Endonuclease reaction
Enzyme:
Restriction enzymes should be kept on ice when they are not in the freezer. The enzyme should always be the last component added to a reaction.
DNA
The preparation of DNA to be cleaved should be free of contaminants such as phenol, chloroform, alcohol, EDTA, detergents, or excessive salts, all of which can interfere with restriction enzyme activity. DNA methylation is also an important element of a restriction ndigest.
Restriction Buffer
We provide a 10X restriction buffers with each restriction enzyme to ensure optimal (100%) activity. The buffer should be used at 1X concentration in the reaction.
Reaction Volume:
By definition, 1µl of restriction enzyme will completely digest 1µg of substrate DNA in a 50µl reaction in 60 minutes. This enzyme - DNA - Reaction volume ratio can be used as guide when designing reactions.
Mixing:
The reaction must be thoroughly mixed to achieve complete digestion. We recommend gently pipetting the reaction mixture up and down or flicking the reaction tube. Follow with a quich spin-down in a microcentrifuge. Do not vortex the reaction.
Incubation Temperature:
The recommended incubation temperature for most restriction enzymes is 37ºC. Restriction enzymes isolated from thermophilic bacteria require higher incubation temperatures ranging from 50ºC to 65ºC. Please refer instruction manual and certificate of analysis' for appropriate incubation temperature.
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