New England Biolabs, R0171S, HpaII

HpaII has been reformulated with Recombinant Albumin (rAlbumin) beginning with Lot #10232250. Related Categories Hydroxymethylation Detection and Analysis,, Methylation Sensitive and Methylation Dependent Restriction Enzymes for Epigenetics,, Restriction Enzymes for Epigenetic Analysis, Applications Fast Cloning: Accelerate your cloning workflows with reagents from NEB,, Restriction Enzyme Digestion Specification Unit Definition One unit is defined as the amount of enzyme required to digest 1 µg of λ DNA in 1 hour at 37°C in a total reaction volume of 50 μl. Reaction Conditions 1X rCutSmart™ Buffer Incubate at 37°C 1X rCutSmart™ Buffer 50 mM Potassium Acetate 20 mM Tris-acetate 10 mM Magnesium Acetate 100 µg/ml Recombinant Albumin (pH 7.9 @ 25°C) Activity in NEBuffers NEBuffer™ r1.1: 100% NEBuffer™ r2.1: 50% NEBuffer™ r3.1: <10% rCutSmart™ Buffer: 100% Diluent Compatibility Diluent A Storage Buffer 10 mM Tris-HCl 50 mM NaCl 1 mM DTT 0.1 mM EDTA 200 µg/ml Recombinant Albumin 50% Glycerol pH 7.4 @ 25°C Heat Inactivation 80°C for 20 minutes Methylation Sensitivity dam methylation: Not Sensitive dcm methylation: Not Sensitive CpG Methylation: Blocked FAQ Q: Is HpaII inhibited by salt? A: HpaII is inhibited by salt concentrations of> 50 mM KCl. Q: Is HpaII affected by methylation? A: HpaII will not cut sites if 2nd Cytosine of the recognition sequence CCGG is methylated by SssI methyltransferase or HpaII Methyltransferase. When the 1st cytosine of the sites have been methylated by MspI methyltransferase, the enzyme will cut 300 times slower than unmethylated DNA and 50 times slower if the DNA is hemi-methylated. McClelland, M., Nelson, M., Raschke, E. (1994). Nucleic Acids Research 22, No. 17, 3640-3659. Q: What is the molecular weight of HpaII? A: The molecular weight is 40kD, as estimated by PAGE. Q: Does spermidine increase HpaII activity? A: It is only needed if there is only one HpaII site present. Q: Does HpaII cleave ssDNA? A: No. Q: What is the activity of HpaII at 25°C? A: HpaII is 50-75% active at 25°C. Q: Do I have to set-up digests with Time-Saver™ qualified enzymes for 5-15 minutes? Can I digest longer? A: NEB's Time-Saver™ enzymes have the benefit of working fast (5-15 minutes), but are also designed and qualified to withstand overnight digestions without degradation of DNA. Q: I tested your restriction enzyme on the substrate DNA recommended by NEB, and it appears to be active, however it does not digest my DNA. What could be the reason? A: The quality and cleanliness of the DNA are major factors that can affect enzyme activity. Restriction Enzymes active in rCutSmart® Buffer, but not as active in NEBuffer r2.1 and/or r3.1 (our higher salt buffers), can be inhibited by salt in the reaction. DNA purification procedures that use spin columns can result in DNA solutions with significant levels of salt that can carry over into the reaction and inhibit enzyme activity. To prevent this, we recommend that the DNA solution added to the reaction be no more than 25% of the total reaction volume. This can be attained by adjusting the total reaction volume accordingly. Q: Are HpaII slow sites found in common vectors? A: There is a "slow site" found in SV40. This DNA has only one cut site. The slow acitvity may be due to the enzyme needing two sites to cut sufficiently. Oller, R.A., Broek, V.W., Conrad, M., Topal, M.D. (1991) Biochemistry 30, p.2543-2549. Q: Can you tell me more about the switch from BSA to Recombinant Albumin (rAlbumin) in NEBuffers? A: NEB is excited to announce that we have switched BSA-containing reaction buffers (NEBuffer 1.1, 2.1, 3.1 and CutSmart® Buffer) to Recombinant Albumin-containing buffers (NEBuffer r1.1, r2.1, r3.1 and rCutSmart™ Buffer). We are also in the process of moving all restriction enzyme formulations to contain rAlbumin. We feel that moving away from animal-containing products is a step in the right direction and are able to offer this enhancement at the same price.