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FAQs for EcoRV-HF™ FAQs for Restriction Endonucleases Technical Reference for Restriction Endonucleases Enzyme Finder NEBcutter NEBuffer Chart Double Digest Finder Isoschizomers DNA Sequences and Maps REBASE NEBuffer 4

Home > Products > Restriction Endonucleases > Restriction Endonucleases > EcoRV-HF™ EcoRV-HF™ New | High-Fidelity Enzymes Catalog # Size Concentration Price Qty   R3195S 4,000 units 20,000 units/ml $53.00 Prices are in US dollars and valid only for US orders. Download: MSDS PDF Recognition Site: isoschizomers | compatible ends | single letter code Source: An E. coli strain that carries the cloned and modified (D19A, E27A) EcoRV gene from the plasmid J62 pLG74 (L.I. Glatman) Reagents Supplied: NEBuffer 4 (10X) Enzyme Properties Activity in NEBuffers: NEBuffer 1: 25% NEBuffer 2: 100% NEBuffer 3: 100% NEBuffer 4: 100% When using a buffer other than the optimal (supplied) NEBuffer, it may be necessary to add more enzyme to achieve complete digestion. Methylation Sensitivity: CpG methylation: Blocked by overlapping Heat Inactivation: 65°C for 20 minutes Survival in a Reaction: Minimum units to digest 1 µg of substrate DNA in 16 hours: 0.50 unit(s) Reaction & Storage Conditions Reaction Conditions: 1X NEBuffer 4 Incubate at 37°C. 1X NEBuffer 4: 20 mM Tris-acetate 50 mM potassium acetate 10 mM Magnesium Acetate 1 mM Dithiothreitol pH 7.9 @ 25°C 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. Concentration: 20,000 units/ml Unit Assay Substrate: λ DNA Storage Conditions: 10 mM Tris-HCl 200 mM NaCl 1 mM DTT 0.1 mM EDTA 200 µg/ml BSA 50% Glycerol Storage Temperature: -20°C Diluent Compatibility: Diluent B Notes General notes: Cleaves to leave a ligatable blunt end in the tetracycline resistance gene of pBR322.  EcoRV-HF can be used in conjunction with NEB's LITMUS™ Vectors to add sticky ends to blunt-ended fragments. Usage notes: EcoRV-HF™ has the same specificity as EcoRV (NEB #R0195), but it has been engineered for reduced star activity. FAQs What does HF refer to following the name of the restriction enzyme? When should I choose the HF version of the enzyme? When is star activity a concern? Why does the HF version of the enzyme have a different recommended buffer than the wild type enzyme? Can the change in buffer preference of the HF enzyme be advantageous? Will the HF enzyme produce elevated star activity when used in a buffer other than the one recommended? What does it mean to be Time-Saver qualified? How is the improvement in fidelity of HF restriction endonucleases quantitated? What is the Fidelity Index (FI)? How does the level of star activity of EcoRV-HF compare to EcoRV? What is the difference between EcoRV-HF and EcoRV? What is the specific activity of EcoRV-HF? Is there a difference in cutting close to the ends between EcoRV-HF and EcoRV? Quality Control for Current Lot Quality control values for a specific lot can be found on the datacard which accompanies each product. Ligation and Re-cutting: After a 10-fold overdigestion with EcoRV-HF™, > 95% of the DNA fragments can be ligated with T4 DNA Ligase (at a 5' termini concentration of 1-2 μM) at 16ºC. Of these ligated fragments, > 95% can be recut with EcoRV-HF™. 16-Hour Incubation: A 50 μl reaction containing 1 μg of DNA and 120 units of EcoRV-HF™ incubated for 16 hours at 37ºC resulted in a DNA pattern free of detectable nuclease degradation as determined by agarose gel electrophoresis. Exonuclease Activity: Incubation of a 50 μl reaction containing 200 units of EcoRV-HF™ with 1 μg of a mixture of single and double-stranded [3H] E. coli DNA (205 cpm/μg) for 4 hours at 37ºC released < 0.1% of the total radioactivity. Endonuclease Activity: Incubation of a 50 μl reaction containing 30 units of EcoRV-HF™ with 1 μg of ΦX174 RF I DNA for 4 hours at 37ºC resulted in < 30% conversion to RFII as determined by agarose gel electrophoresis. Reagents Sold Separately NEBuffer 4