This will save you time and ensure consistency across the reactions. Aliquot your DNA into individual tubes and then add the appropriate amount of Master Mix to each tube. If you are digesting a large number of plasmids with the same enzyme(s) (for instance, in a diagnostic digest), you can create a "Master Mix" consisting of all of the reaction components except for the DNA.Learn more at NEB's website about star activity. Examples of nuclease contamination studies for some of. This is due to "Star Activity" and can happen for a variety of reasons, including high glycerol concentration. NEB extensively performs quality controls on all standard and high-fidelity (HF) restriction enzymes.
Sometimes enzymes cut sequences which are similar, but not identical, to their recognition sites. Engineered with performance in mind, HF restriction enzymes are fully active under a broader range of conditions, minimizing off-target products, while offering flexibility in experimental design.See NEB's table of methylation sensitive restriction sites. Plasmids grown in Dam or Dcm methylase positive strains will be resistant to cleavage at certain restriction sites. If your enzyme did not cut, check to make sure that it isn't methylation sensitive.CIP (calf alkaline phosphatase) or SAP (shrimp alkaline phosphatase) are commonly used. Bioz Stars score: 96/100, based on 1 PubMed citations. If you are using blunt ends or a single enzyme to cut the vector, you will need to use a phosphatase to prevent re-circularization of the vector if you are cloning in an insert. New England Biolabs single cutting restriction noti hf enzyme Single Cutting Restriction Noti Hf Enzyme, supplied by New England Biolabs, used in various techniques.Use T4 DNA Polymerase or Klenow DNA Polymerase I for 3′ overhang removal and 5′ overhang fill-in. If you cannot find compatible sticky ends, you will need to fill in the overhangs and conduct a blunt end ligation.See NEB's compatible cohesive ends chart.
coli DH5 alpha cells and selected on agar plates with selective antibiotic marker. High Fidelity (HF) Restriction Enzymes have 100 activity in rCutSmart Buffer single-buffer simplicity means more straightforward and streamlined sample.
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