R0546 FAQs for TfiI, Restriction Endonucleases, Intl, NEB
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TfiI FAQ

Q1: The NEB catalog has historically stated that activity in NEBuffer 4 is less than 100% yet this enzyme is now supplied with NEBuffer 4. What have you changed?
Q2: Has the conversion to NEBuffer 4 altered any of the properties of the restriction enzyme?
Q3: If I have an old tube of enzyme, what NEBuffer should I use?
Q4: Will the new enzyme work in the originally supplied NEBuffer?
Q5: Why is NEB switching this restriction enzyme to NEBuffer 4?
Q6: What is Star Activity and how can it be avoided?
Q7: Do degenerate recognition sites need to be palindromic?
Q8: How can this enzyme be inactivated?

Q1: The NEB catalog has historically stated that activity in NEBuffer 4 is less than 100% yet this enzyme is now supplied with NEBuffer 4. What have you changed?

A1: All our restriction enzymes were re-assayed in both NEBuffer 4 vs. the previously supplied NEBuffer. In a few cases minor formulation changes were required to bring the activity to 100% in NEBuffer 4.


Q2: Has the conversion to NEBuffer 4 altered any of the properties of the restriction enzyme?

A2: In general the properties of the restriction enzyme remain the same although for some enzymes, some minor changes in heat inactivation, Time-Saver™ qualification, etc. were observed. All the updated information can be found on the supplied data card as well as at
www.neb.com.


Q3: If I have an old tube of enzyme, what NEBuffer should I use?

A3: Our restriction enzymes are color coded on the label for the appropriate NEBuffer and can either be used with the previously supplied NEBuffer or with NEBuffer 4. To ensure 100% cleavage in NEBuffer 4, additional units of enzyme and/or longer incubation time may be necessary.


Q4: Will the new enzyme work in the originally supplied NEBuffer?

A4: Yes it will. In all instances the restriction enzyme will have 100% activity in the originally supplied NEBuffer.


Q5: Why is NEB switching this restriction enzyme to NEBuffer 4?

A5: Our main goal is to simplify the NEBuffer system so that the majority of restriction enzymes are compatible in a single buffer. We now supply 162 restriction enzymes with NEBuffer 4 including all the new High Fidelity (HF) restriction enzymes.


Q6: What is Star Activity and how can it be avoided?

A6: It has been demonstrated that under extreme non-standard conditions, restriction endonucleases are capable of cleaving sequences which are similar but not identical to their defined recognition sequence. This altered or relaxed specificity has been termed "star" activity. It has been suggested that star activity may be a general property of restriction endonucleases and that any restriction endonuclease can be made to cleave noncanonical sites under certain extreme conditions.

The manner in which an enzyme's specificity is altered depends on the enzyme and on the conditions employed to induce the 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.

Star activity is completely controllable in the vast majority of cases and is generally not a concern when performing restriction endonuclease digests. New England Biolabs' enzymes will not exhibit star activity when used under recommended conditions in their supplied NEBuffers. Listed below are reaction conditions known to induce or inhibit star activity.

Conditions that Contribute to Star Activity
1. High glycerol concentration [> 5% v/v]
2. High units to g of DNA ratio [Varies with each enzyme, usually >100 units/g]
3. Low ionic strength [< 25 mM]
4. High pH [> pH 8.0]
5. Presence of organic solvents [DMSO, ethanol, ethylene glycol, dimethylacetamide, dimethylformamide, sulphalane]
6. Substitution of Mg++ with other divalent cations [Mn++, Cu++, Co++, Zn++]

Inhibiting Star Activity
If you are concerned about star activity, we recommend the following guidelines.
1. Use as few units as possible to get a complete digestion. This avoids overdigestion and reduces the final glycerol concentration in the reaction.
2. Make sure the reaction is free of any organic solvents such as alcohols which might be present in the DNA preparation.
3. Raise the ionic strength of the reaction buffer to 100?150 mM (provided the enzyme is not inhibited by high salt).
4. Lower the pH of the reaction buffer to pH 7.0.
5. Use Mg++ as the divalent cation.


Q7: Do degenerate recognition sites need to be palindromic?

A7: Most restriction enzyme recognition sites are palindromic and include only specified base pairs (i.e., EcoRI recognizes GAATTC). However, some enzymes have degenerate sites, meaning that they contain one or more base pairs that are not specifically defined (i.e., BsrFI recognizes RCCGGY, where R= A or G and Y= C or T). For degenerate enzymes, any base represented by the single letter code may be present at either location in the recognition site for cleavage to occur. For example, BsrFI recognizes all of the following sequences: ACCGGC, ACCGGT, GCCGGC, GCCGGT.


Q8: How can this enzyme be inactivated?

A8: To inactivate enzymes that cannot be heat killed, we recommend a phenol extraction followed by ethanol precipitation or a commercial spin column designed to purify DNA.