NEB CutSmart Buffer:
CutSmart = Clone Smart!
Over 200 restriction enzymes are 100% active in a single buffer, CutSmart Buffer, making it significantly easier to set up your double digest reactions. Since CutSmart Buffer includes BSA, there are also fewer tubes and pipetting steps to worry about. Additionally, many DNA modifying enzymes are 100% active in CutSmart Buffer, eliminating the need for subsequent purification.
How is NEB’s new buffer system going to help me?
Although the old buffer system worked well, NEB is continuously looking for ways to enhance the convenience and performance of its products for its customers. By adding BSA to the buffer, we were able to offer > 200 enzymes that cut in a single buffer. This improves ease-of-use, especially when performing double digests. It also eliminates the need to add BSA separately.
Q. Has the conversion to the new buffer system altered any of the properties of the Restriction Enzyme itself?
A. The nature of the restriction enzymes is the same. However, for a handful of enzymes, BSA enhances activity. As a result, to keep the enzyme titer within specification for these products, the protein concentration may have been adjusted slightly.
Q. If I have an old tube of Restriction Enzyme, what NEBuffer should I use?
A. All NEB Restriction Enzymes have color-coded labels for the appropriate NEBuffer; this system can either be used with the previously supplied NEBuffer or with the newly recommended buffer. We have maintained the color-coding of our buffer and enzyme tubes so you can always match an enzyme to its recommended buffer.
Q. I currently have an old tube of Restriction Enzyme – is it still active in the new buffer?
A. Provided it is still prior to the product’s expiry date, yes. The new buffers are mostly identical, except that BSA has been added directly to the buffer and DTT has been removed. BSA will not harm the reaction and may even enhance it in some cases. Extensive testing has shown that DTT is not required.
Q. I currently have an old tube of NEBuffer – can I still use it or should I throw it away?
A. You can use the old tube of NEBuffer until the expiration date. Please remember to follow the original recommendations for use with the old buffer, which may include the addition of BSA for some enzymes.
Q. My enzyme used to come with another NEBuffer, but CutSmart Buffer is now recommended? Why?
A. Reformulation of the NEB buffers has allowed some restriction enzymes to now also work well in CutSmart buffer. This enabled us to offer >200 restriction enzymes in a single buffer, greatly increasing convenience, especially for double digests.
Q. Why aren’t all your enzymes available in CutSmart Buffer?
A. Some restriction enzymes require high salt and CutSmart Buffer is a low salt buffer. Over 200 restriction enzymes are supplied with CutSmart Buffer, but the remaining enzymes performed more optimally in another buffer. Please refer to the Performance Chart.
Q. How do I perform a double digest with an enzyme that comes with the new buffer and an enzyme that comes with the old buffer system?
A. We recommend using the Double Digest Finder and use the buffer it recommends. For example, you have old buffer NEBuffer 1 with one of the enzymes and CutSmart Buffer with the second enzyme. If Double Digest Finder recommends to use NEBuffer 1.1, then you can use NEBuffer 1 + BSA for the double digest. Alternatively, if it recommends CutSmart Buffer, then you can simply use the new buffer.
Q. My restriction enzyme used to be available at a lower concentration. Why does it now come at a higher concentration of 10,000 u/ml?
A. NEB has standardized many of its restriction enzymes to 10,000 u/ml. This contributes to ease of use and often results in improved performance by having less glycerol in the digest.
Q. Why did you add BSA into all the restriction enzyme reaction buffers?
A. As an added convenience for our customers, NEB has included BSA in all its restriction enzyme buffers, eliminating the need to add it as a separate step. This makes digestion reactions simpler, set-up time even faster, and decreases the number of tubes to store.
Q. Why did you remove DTT from your restriction enzyme buffers?
A. DTT was originally included in the buffer formulation for historical reasons. That is, DTT is commonly used is many molecular biology enzyme buffers and thus was also included in these restriction enzyme buffers, when they were first developed. However, after extensive testing, we have found that it is not necessary for restriction enzyme activity. In addition, BSA in the presence of DTT can on occasions form a white precipitate after repeated freeze/thaws.
Q. I don’t want to use BSA in my buffer. What are my options?
A. NEB continues to offer the old buffers, which do not contain BSA in their formulation, and thus can be purchased separately. Please note that BSA has always been included in the formulation of the enzymes’ storage buffer.
Q. How do NEB modifying enzymes perform in CutSmart™ Buffer?
A. NEB has tested many modifying enzymes involved in the cloning workflow and has confirmed most have 100% functional activity in CutSmart™ Buffer. Please refer to the “Activity of DNA Modifying Enzymes in CutSmart™ Buffer” for specific information.
Q. My enzyme is no longer Time-Saver™ qualified. What happened?
A. The Time-Saver™ definition originally referred to any restriction enzyme that could cut substrate DNA rapidly (in 5-15 minutes). We have recently made the definition more stringent; Time-Saver qualified enzymes can also safely be used overnight without any negative effects. If your enzyme was Time-Saver qualified, but no longer is, it will still cut DNA in 5-15 minutes. However, it may not be able to be incubated overnight and is usually accompanied by a note explaining why. A full list of enzymes that digest DNA in 5-15 minutes, as well as Time-Saver qualified enzymes, can be found at www.neb-online.de.
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.