New England Biolabs, B8534S, Diluent C (with rAlbumin)

Related Categories Restriction Endonuclease Buffers & Diluents Applications Restriction Enzyme Digestion Specification 1X Buffer Components 250 mM NaCl 10 mM Tris-HCl 1 mM DTT 0.1 mM EDTA 0.15% Triton® X-100 200 µg/ml Recombinant Albumin 50% Glycerol pH 7.4@25°C FAQ Q: How long can the diluted enzyme be stored? A: The stability of each enzyme diluted below selling concentration has not been tested. Many enzymes will remain active for long periods of time when stored diluted at -20°C. Use a control substrate to test dilutions that have been stored. Q: Why is my Restriction Enzyme not cutting DNA? A: There can be several different reasons why your restriction enzyme does not cut the DNA as reviewed in this video. 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. Monarch Nucleic Acid Purification Kits may be used to purify sample DNA prior to digestion. If you are having difficulty cleaving your DNA substrate, we recommend the following control reaction: Incubate experimental DNA in reaction buffer without restriction enzyme (degradation of DNA indicates contamination in the DNA preparation or reaction buffer) and control DNA (DNA with multiple known sites for the enzyme, e.g. lambda DNA) with restriction enzyme to more accurately judge whether or not the reaction went to completion. 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. Additional troubleshooting help is also available. DNA methylation is also an important element of a restriction digest. If methylation inhibition is suspected, please refer to the link describing dam, dcm and CpG methylation. Q: Why do I see additional DNA bands on my gel after a restriction digest? A: There can be a few different reasons why you observe additional bands in your digest. For a discussion on this topic please refer to the video above. Typically, off-target DNA bands are caused by either partial digestion or Star Activity. You need to compare your digestion to the expected DNA banding pattern. If the bands in both lanes are similar to the expected pattern and the additional bands are limited to spaces within the upper and lower bands of the expected pattern, the digestion is incomplete (partial). In this case, you may need to purify the DNA to remove any contaminants, use more enzyme and/or increase the incubation time to ensure complete digestion. If the additional bands are also seen below the lowest band of the expected pattern, and expected bands are being cut, the digestion is likely to be exhibiting Star activity. Your reactions conditions are driving the enzyme to cleave additional, non-canonical DNA sequences. You should repeat the digestion under one, or more, of the following modified reactions conditions: ∙ Decrease the amount of enzyme ∙ Decrease the incubation time ∙ Increase the reaction volume or ∙ Use an appropriate NEB High Fidelity (HFTM) restriction enzyme Q: How many nucleotides do I have to add adjacent to the RE recognition site in order to get efficient cutting? A: Restriction Enzyme Digest Protocol: Cutting Close to DNA End