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4 TITLE 4 TIPS & TRICKS 4 The NEBuffer System For optimal restriction enzyme digests NEB provides a colour-coded 10X NEBuffer with each restriction enzyme to ensure optimal (100%) activity. Most of our enzymes are supplied with one of our four standard NEBuffers. Occasionally, an enzyme has specific buffer requirements not met by one of the four standard NEBuffers, in which case the enzyme is supplied with its own unique NEBuffer. As part of our ongoing committment to research, we are constantly looking for ways to improve the production and efficiency of restriction enzymes. This may result in changes to buffer requirements which has been the case recently for one of our most commonly used enzymes: BamHI. BamHI B r C 3 b 6 n p Cleaving a DNA substrate with two restriction endonucleases simultaneously (double digestion) is a common timesaving procedure. Selecting the best NEBuffer to provide reaction conditions that optimize enzyme activity as well as avoid star activity associated with some enzymes is an important consideration. Each enzyme is supplied with its optimal NEBuffer to ensure 100% activity. NEBuffer compositions are listed on page 278 of our current catalogue and on the data card sent with each enzyme. The Activity Chart for Restriction Enzymes (see below to request your free poster) rates the percentage activity of each restriction endonuclease in the four standard NEBuffers. The NEBuffer that results in the most activity for both enzymes should be used for the double digest as long as star activity is not a factor. Depending on an enzyme's activity rating in a non-optimal NEBuffer, the number of units of enzyme or incubation time of the reaction may be adjusted to compensate for a slower rate of cleavage. If no single NEBuffer can be found to satisfy the buffer requirements of both enzymes, the reactions can be done sequentially. First, cleave with the restriction endonuclease that requires the lower salt reaction conditions, then adjust the salt concentration of the reaction (using a small volume of a concentrated salt solution) to approximate The New and Updated NEBuffer Poster BamHI is now supplied with NEBuffer 3 instead of a Unique Buffer following improvements to the production of this enzyme Setting up a Successful Double Digest the reaction conditions of the second restriction endonuclease. Add the second enzyme and incubate to complete the second reaction. Alternatively, a spin column can be used to isolate the DNA prior to the second reaction. Performing double digests with enzymes that are supplied with their own unique NEBuffer and not one of the four standard NEBuffers is also simple. In many cases, double digests using any of these enzymes with an enzyme that is supplied with one of the four standard NEBuffers can be done in the unique NEBuffer. This will ensure that the enzyme with the more specific buffer requirements will work optimally. See page 284 of our current catalogue for the most commonly used restriction endonucleases’ activity ratings in unique buffers. When using restriction endonucleases in non-optimal NEBuffers, more enzyme or longer digestion time may be needed to compensate for the slower rate of cleavage under those conditions. Check the Activity Chart for Restriction Enzymes to see how well the second enzyme performs in the salt range of the unique NEBuffer. NEBuffer 1 2 3 4 % Activity 75 100 100 100 Double Digest Tips: If BSA is a buffer requirement for either enzyme, add it to the double digest reaction. BSA will not inhibit any restriction enzyme. The final concentration of glycerol in any reaction should be less than 5% to minimize the possibility of star activity. All our restriction enzymes are supplied in 50% glycerol so the total volume of enzyme in a reaction should be a maximum of 10% of the total reaction volume. An increase in total reaction volume may be necessary. Double digestion is not recommended for certain enzyme combinations. In these cases a sequential digest is required. These combinations are indicated by the abbreviation "seq" in the Double Digest table on the buffer poster. Our data is based on 1–2 hour digests. Overnight double digests should be avoided due to possible star activity. BsgI requires SAM. SAM as an additive does not have a negative effect on the activity of other enzymes. For help choosing double digest conditions try Double Digest Finder at www.neb.uk.com This web-based tool can be used to suggest conditions for double digestion using any two NEB restriction enzymes. The NEBuffer chart helps you select the best conditions for double digests, shows the optimal (supplied) NEBuffer and approximate activity in the four standard NEBuffers for each enzyme. To request a copy please email us: bufferposter@uk.neb.com or use our online literature request form at: www.neb.uk.com
High Fidelity (HF) Restriction Enzymes The latest innovation in restriction enzyme technology As part of our ongoing commitment to the study and improvement of restriction enzymes, we are pleased to introduce a line of High Fidelity (HF) restriction enzymes. These engineered enzymes have the same specificity as their established counterparts, however certain properties have been altered, including buffer requirements and enzyme fidelity. These modifications provide more flexibility in setting up restriction enzyme digests. The overall goal of engineering restriction enzymes is to provide improved enzymes that will allow more flexibility with respect to reaction volume, incubation time and buffer compatibility. Each of these enzymes has been purified to the same high standards as our other restriction enzymes, and are available at the same low price. The introductory selection of engineered restriction enzymes offers the benefit of reduced star activity. Star activity, or relaxed specificity, is an intrinsic property of restriction enzymes. Most restriction enzymes will not exhibit star activity when used under the recommended reaction conditions. However, for enzymes that have reported star activity, extra caution must be taken to set up reactions under the recommended conditions to avoid unwanted cleavage. Different techniques such as cloning, genotyping, mutational analysis, mapping, probe preparation, sequencing and methylation detection employ a wide range of reaction conditions and require the use of enzymes under suboptimal conditions. These new high fidelity (HF) enzymes will offer increased flexibility to reaction setup, maximizing results under a wider range of conditions. In addition to reduced star activity, all of these engineered enzymes work optimally in NEBuffer 4, which has the the highest level of enzymes compatibility, and may simplify double digest reactions. They are also Time-Saver qualified, and will digest substrate DNA in five minutes. In order to distinguish these engineered enzymes, the letters –HF have been added to the restriction enzyme name. An icon ( E ) designating that the enzyme has been engineered will appear with the product entry, on the datacard and on the website. In addition, icons for the enhanced properties (such as reduced star activity, R ) that these new enzymes possess will also be included. These enzymes will be packaged with a purple cap to distinguish them from our existing restriction enzymes. Five HF enzymes are currently available with more to follow. Ordering Information: NheI-HF E R #R3131S 1,000 units 20,000 units/ml £44 PvuII-HF E R #R3151S 5,000 units 20,000 units/ml £45 SalI-HF E R #R3138S 2,000 units 20,000 units/ml £40 ScaI-HF E R #R3122S 1,000 units 20,000 units/ml £45 SphI-HF E R #R3182S 500 units 20,000 units/ml £48 same low price as standard enzymes ScaI-HF M 4 3 4 NEBuffer ScaI ScaI is one of the most frequently reported enzymes to exhibit star activity (unwanted cleavage). While star activity is observed with ScaI in NEBuffer 3 (supplied buffer) as well as NEBuffer 4, star activity is significantly reduced with ScaI-HF. 20 μl reactions were set up containing 2 μl of enzyme and incubated for 1 hour. Conditions were designed to simulate those obtained when a double digest is performed in a 20 μl reaction. Marker M is the 1 kb DNA Ladder (NEB #N3232). New England Biolabs ... for the highest quality Restriction Enzymes. Manufactured and quality controlled by NEB – our restriction enzymes aren’t made by anyone else. * * * * NEW unwanted cleavage TITLE 5 PROTEIN LATEST NEWS: TOOLSRESTRICTION ENZYMES 3 5 5
Page 1 and 2: NEB UK Update Spring 2008 NEW ENGLA
Page 3: ColorPlus Prestained Protein Marker
Page 7 and 8: High Performance PCR THE INTEGRATED
Page 9 and 10: SPECIAL OFFER CST Lysis Buffers, Ma
Page 11 and 12: Light chain 3 (LC3), which can serv

NEB UK - New England Biolabs (UK)

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