{"id":1844,"date":"2015-04-24T09:05:44","date_gmt":"2015-04-24T09:05:44","guid":{"rendered":"http:\/\/www.neb-online.fr\/?page_id=1844"},"modified":"2022-11-14T14:35:13","modified_gmt":"2022-11-14T13:35:13","slug":"hf-enzymes","status":"publish","type":"page","link":"https:\/\/www.neb-online.fr\/en\/cloning-synthetic-biology\/restrictionsenzyme\/hf-enzymes\/","title":{"rendered":"HF Enzymes"},"content":{"rendered":"

[vc_row][vc_column css=”.vc_custom_1429123657387{margin-bottom: 20px !important;}”][vc_column_text]Engineered for performance under a wide range of conditions <\/strong>[\/vc_column_text][vc_column_text]For over 35 years, New England Biolabs has been developing innovative solutions for molecular biology applications. The respected leader in the field of restriction enzyme biology, NEB has developed a line of High-Fidelity (HF\u00ae<\/sup>) Restriction Enzymes. These engineered enzymes have the same specificity as the native enzyme, with the added benefit of reduced star activity, rapid digestion (5-15 minutes) and 100% activity in CutSmart\u00ae<\/sup> Buffer<\/a>.<\/p>\n

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.<\/p>\n

Optimized performance for a wide range of conditions <\/strong><\/p>\n

High Fidelity Restriction Enzymes have been engineered by exchanging functional amino acid residues and then screening for optimal mutants that perform under a wide range of conditions. Whether you are setting up digests for 5-15 minutes or overnight, or using varying amounts of enzymes, HF enzymes ensure the performance you need.[\/vc_column_text][vc_row_inner][vc_column_inner width=”3\/4″][vc_column_text]<\/p>\n

Advantages<\/h2>\n

[\/vc_column_text][vc_column_text]\"neb_reduced-star_icon2\"<\/p>\n

Reduced star activity eliminates unwanted cleavage<\/strong>[\/vc_column_text][vc_column_text]\"NEB_CutSmart_icon\"<\/p>\n

One buffer convenience<\/a> with no loss of performance<\/strong>[\/vc_column_text][vc_column_text]\"neb_timesaver_icon2\"<\/p>\n

Time-Saver<\/a> qualified for 5-15 minute digests, flexible enough to digest overnight.<\/strong>[\/vc_column_text][vc_column_text]\"value_icon\"Added flexibility without added cost<\/strong>[\/vc_column_text][vc_column_text]\"value_icon\"Including 1 ml Gel Loading Dye, Purple (6X) gratis<\/a>[\/vc_column_text][\/vc_column_inner][vc_column_inner width=”1\/4″][vc_single_image image=”1141″ img_size=”full” alignment=”center”][\/vc_column_inner][\/vc_row_inner][\/vc_column][\/vc_row][vc_row][vc_column][vc_separator color=”custom” accent_color=”#919191″][\/vc_column][\/vc_row][vc_row][vc_column][vc_column_text]<\/p>\n

Available HF Enzymes<\/h2>\n\n\n\n
\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

PRODUCT<\/span><\/strong><\/p>\n<\/th>\n

Ref.<\/span><\/th>\n<\/tr>\n
AgeI-HF\u00ae<\/sup><\/strong><\/a><\/span><\/td>\nR3552<\/span><\/strong><\/td>\n<\/tr>\n
ApoI-HF\u00ae<\/sup><\/strong><\/a><\/span><\/td>\nR3566<\/span><\/strong><\/td>\n<\/tr>\n
BamHI-HF\u00ae<\/sup><\/strong><\/a><\/span><\/td>\nR3136<\/span><\/strong><\/td>\n<\/tr>\n
BbsI-HF\u00ae<\/sup><\/strong><\/a><\/span><\/td>\nR3539<\/span><\/strong><\/td>\n<\/tr>\n
BclI-HF\u00ae<\/sup><\/strong><\/a><\/span><\/td>\nR3160<\/span><\/strong><\/td>\n<\/tr>\n
BmtI-HF\u00ae<\/sup><\/strong><\/a><\/strong><\/span><\/td>\nR3658<\/span><\/strong><\/td>\n<\/tr>\n
BsaI-HF<\/strong>\u00ae<\/sup> V2<\/strong><\/span><\/a><\/td>\nR3733<\/span><\/strong><\/td>\n<\/tr>\n
BsiWI-HF\u00ae<\/sup><\/strong><\/a><\/span><\/td>\nR3553<\/span><\/strong><\/td>\n<\/tr>\n
BsrGI-HF\u00ae<\/sup><\/strong><\/a><\/span><\/td>\nR3575<\/span><\/strong><\/td>\n<\/tr>\n
BstEII-HF\u00ae<\/sup><\/strong><\/a><\/strong><\/span><\/td>\nR3162<\/span><\/strong><\/td>\n<\/tr>\n
BstZ17I-HF\u00ae<\/sup><\/strong><\/a><\/strong><\/span><\/td>\nR3162<\/span><\/strong><\/td>\n<\/tr>\n
DraIII-HF\u00ae<\/sup><\/strong><\/a><\/span><\/td>\nR3510<\/span><\/strong><\/td>\n<\/tr>\n
EagI-HF\u00ae<\/sup><\/strong><\/a><\/span><\/td>\nR3505<\/span><\/strong><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<\/td>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

PRODUCT<\/span><\/strong><\/p>\n<\/th>\n

Ref.<\/span><\/th>\n<\/tr>\n
EcoRI-HF\u00ae<\/sup><\/strong><\/a><\/span><\/td>\nR3101<\/span><\/strong><\/td>\n<\/tr>\n
EcoRV-HF\u00ae<\/sup><\/strong><\/a><\/span><\/td>\nR3195<\/span><\/strong><\/td>\n<\/tr>\n
HindIII-HF\u00ae<\/sup><\/strong><\/a><\/span><\/td>\nR3104<\/span><\/strong><\/td>\n<\/tr>\n
KpnI-HF\u00ae<\/sup><\/strong><\/a><\/span><\/td>\nR3142<\/strong><\/span><\/td>\n<\/tr>\n
MfeI-HF\u00ae<\/sup><\/strong><\/a><\/span><\/td>\nR3589<\/strong><\/span><\/td>\n<\/tr>\n
MluI-HF\u00ae<\/sup><\/strong><\/a><\/span><\/td>\nR3198<\/strong><\/span><\/td>\n<\/tr>\n
NcoI-HF\u00ae<\/sup><\/strong><\/a><\/span><\/td>\nR3193<\/strong><\/span><\/td>\n<\/tr>\n
NheI-HF\u00ae<\/sup><\/strong><\/a><\/span><\/td>\nR3131<\/strong><\/span><\/td>\n<\/tr>\n
NotI-HF\u00ae<\/sup><\/strong><\/a><\/span><\/td>\nR3189<\/strong><\/span><\/td>\n<\/tr>\n
NruI-HF\u00ae<\/sup><\/strong><\/a><\/span><\/td>\nR3192<\/strong><\/span><\/td>\n<\/tr>\n
NsiI-HF\u00ae<\/sup><\/strong><\/a><\/span><\/td>\nR3127<\/strong><\/span><\/td>\n<\/tr>\n
PstI-HF\u00ae<\/sup><\/strong><\/a><\/span><\/td>\nR3140<\/strong><\/span><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<\/td>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

PRODUCT<\/span><\/strong><\/p>\n<\/th>\n

Ref.<\/span><\/th>\n<\/tr>\n
PvuI-HF\u00ae<\/sup><\/strong><\/a><\/span><\/td>\nR3150<\/strong><\/span><\/td>\n<\/tr>\n
PvuII-HF\u00ae<\/sup><\/strong><\/a><\/span><\/td>\nR3151<\/strong><\/span><\/td>\n<\/tr>\n
SacI-HF\u00ae<\/sup><\/strong><\/a><\/span><\/td>\nR3156<\/strong><\/span><\/td>\n<\/tr>\n
SalI-HF\u00ae<\/sup><\/strong><\/a><\/span><\/td>\nR3138<\/strong><\/span><\/td>\n<\/tr>\n
SbfI-HF\u00ae<\/sup><\/strong><\/a><\/span><\/td>\nR3642<\/strong><\/span><\/td>\n<\/tr>\n
ScaI-HF\u00ae<\/sup><\/strong><\/a><\/span><\/td>\nR3122<\/strong><\/span><\/td>\n<\/tr>\n
SpeI-HF\u00ae<\/sup><\/strong><\/a><\/span><\/td>\nR3133<\/span><\/strong><\/td>\n<\/tr>\n
SphI-HF\u00ae<\/sup><\/strong><\/a><\/span><\/td>\nR3182<\/span><\/strong><\/td>\n<\/tr>\n
SspI-HF\u00ae<\/sup><\/strong><\/a><\/span><\/td>\nR3132<\/span><\/strong><\/td>\n<\/tr>\n
StyI-HF\u00ae<\/sup><\/strong><\/a><\/span><\/td>\nR3500<\/span><\/strong><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

[\/vc_column_text][\/vc_column][\/vc_row][vc_row][vc_column][vc_separator color=”custom” accent_color=”#919191″][\/vc_column][\/vc_row][vc_row][vc_column width=”2\/3″][vc_column_text]<\/p>\n

Star Activity<\/h2>\n

Under non-standard reaction conditions, some restriction enzymes are capable of cleaving sequences which are similar, but not identical, to their defined recognition sequence. This altered specificity has been termed \u201cstar activity”. It has been suggested that star activity is a general property of restriction endonucleases (1) and that any restriction endonuclease will cleave noncanonical sites under certain extreme conditions, some of which are listed below. Although the propensity for star activity varies, the vast majority of enzymes from New England Biolabs will not exhibit star activity when used under recommended conditions in their supplied NEBuffers. If an enzyme has been reported to exhibit star activity, it will be indicated in the product entry found in the catalog, on the datacard or on our web site.<\/p>\n

The manner in which an enzyme’s specificity is altered depends on the enzyme and the reaction conditions which induce star activity. The most common types of altered activity are single base substitutions, truncation of the outer bases in the recognition sequence, and single-strand nicking (2). Some enzymes exhibit relaxation of sequence specificity under standard conditions and in the presence of the cognate site are capable of cleaving non-cognate (secondary) sites (3).[\/vc_column_text][\/vc_column][vc_column width=”1\/3″][vc_column_text]\"Flexible_Enzyme_Gel_M\" EcoRI-HF\u00ae (NEB #R3101<\/a>) shows no star activity in overnight digests, even when used at higher concentrations. 50 \u03bcl reactions were set up using 1 \u03bcg of Lambda DNA, the indicated amount of enzyme and the recommended reaction buffer. Reactions were incubated overnight at 37\u00b0C. Marker M is the 1 kb DNA Ladder (NEB# N3232<\/a>)<\/span>[\/vc_column_text][\/vc_column][\/vc_row][vc_row][vc_column][vc_column_text]<\/p>\n

How to avoid Star Activity<\/h3>\n

[\/vc_column_text][vc_column_text css=”.vc_custom_1429866694882{margin-top: -20px !important;}”]<\/p>\n\n\n\n\n\n\n\n\n\n\n
Conditions that Contribute to Star Activity<\/th>\nSteps that can be Taken to Inhibit Star Activity<\/th>\n<\/tr>\n<\/thead>\n
High glycerol concentration (> 5% v\/v)<\/td>\nRestriction enzymes are stored in 50% glycerol, therefore the amount of enzyme added should not exceed 10% of the total reaction volume. Use the standard 50 \u00b5l reaction volume to reduce evaporation during incubation.<\/td>\n<\/tr>\n
High concentration of enzyme\/\u00b5g of DNA ratio (varies with each enzyme, usually 100 units\/\u00b5g)<\/td>\nUse the fewest units possible to achieve digestion. This avoids overdigestion and reduces the final glycerol concentration in the reaction.<\/td>\n<\/tr>\n
Non-optimal buffer<\/td>\nWhenever possible, set up reactions in the recommended buffer. Buffers with differing ionic strength and pH may contribute to star activity.<\/td>\n<\/tr>\n
Prolonged reaction time<\/td>\nUse the minimum reaction time required for complete digestion. Prolonged incubation may result in increased star activity, as well as evaporation.<\/td>\n<\/tr>\n
Presence of organic solvents [DMSO, ethanol (4), ethylene glycol, dimethylacetamide, dimethylformamide, sulphalane (5)]<\/td>\nMake sure the reaction is free of any organic solvents, such as alcohols, which might be present in the DNA preparation.<\/td>\n<\/tr>\n
Substitution of Mg2+<\/sup> with other divalent cations (Mn2+<\/sup>, Cu2+<\/sup>, Co2+<\/sup>, Zn2+<\/sup>)<\/td>\nUse Mg2+<\/sup> as the divalent cation. Other divalent cations may not fit correctly into the active site of the restriction enzyme, possibly interfering with proper recognition.<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

[\/vc_column_text][vc_column_text]References:<\/p>\n

1. Nasri, M. and Thomas, D. (1986) Nucleic Acids Res<\/em>. 14, 811. PMID: 3003698
\n<\/a>2. Barany, F. (1988) Gene<\/em> 65, 149. PMID: 2842230
\n<\/a>3. Bitinaite, J. and Schildkraut, I. (2002) Proc. Natl. Acad. Sci USA<\/em> 99, 1164-1169. PMID: 11818524
\n<\/a>4. Nasri, M. and Thomas, D. (1987) Nucleic Acids Res<\/em>. 15, 7677. PMID: 2823216
\n<\/a>5. Tikchinenko, T. I. et al. (1978) Gene.<\/em> 4, 195-212. PMID: 33871<\/a>[\/vc_column_text][\/vc_column][\/vc_row]<\/p>\n<\/div>","protected":false},"excerpt":{"rendered":"

[vc_row][vc_column css=”.vc_custom_1429123657387{margin-bottom: 20px !important;}”][vc_column_text]Engineered for performance under a wide range of conditions [\/vc_column_text][vc_column_text]For over 35 years, New England Biolabs has been developing innovative solutions for molecular biology applications. The respected leader in the field of restriction enzyme biology, NEB has developed a line of High-Fidelity (HF\u00ae) Restriction Enzymes. These engineered enzymes have the same specificity… Read more »<\/a><\/p>\n","protected":false},"author":15,"featured_media":1849,"parent":1830,"menu_order":0,"comment_status":"open","ping_status":"open","template":"page-sidebar-cloning-synthetic-biology.php","meta":{"footnotes":""},"class_list":["post-1844","page","type-page","status-publish","has-post-thumbnail","hentry"],"yoast_head":"\nHF Enzymes - New England Biolabs France<\/title>\n<meta name=\"robots\" content=\"index, follow, max-snippet:-1, max-image-preview:large, max-video-preview:-1\" \/>\n<link rel=\"canonical\" href=\"https:\/\/www.neb-online.fr\/en\/cloning-synthetic-biology\/restrictionsenzyme\/hf-enzymes\/\" \/>\n<meta property=\"og:locale\" content=\"en_US\" \/>\n<meta property=\"og:type\" content=\"article\" \/>\n<meta property=\"og:title\" content=\"HF Enzymes - New England Biolabs France\" \/>\n<meta property=\"og:description\" content=\"[vc_row][vc_column css=”.vc_custom_1429123657387{margin-bottom: 20px !important;}”][vc_column_text]Engineered for performance under a wide range of conditions [\/vc_column_text][vc_column_text]For over 35 years, New England Biolabs has been developing innovative solutions for molecular biology applications. The respected leader in the field of restriction enzyme biology, NEB has developed a line of High-Fidelity (HF\u00ae) Restriction Enzymes. These engineered enzymes have the same specificity... 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