A1: XbaI is blocked by overlapping dam methylation. If the recognition site is preceded by GA or followed by TC dam methylase GATC will overlap the XbaI site TˆCTAGA: blocked are ga TˆCTAGA and TˆCTAGAtc. To remove dam methylation use a dam deficient strain like GM2163 (E4105S).
Q2: 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?
A2: 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.
Q3: Has the conversion to NEBuffer 4 altered any of the properties of the restriction enzyme?
A3: 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.
Q4: If I have an old tube of enzyme, what NEBuffer should I use?
A4: 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.
Q5: Will the new enzyme work in the originally supplied NEBuffer?
A5: Yes it will. In all instances the restriction enzyme will have 100% activity in the originally supplied NEBuffer.
Q6: Why is NEB switching this restriction enzyme to NEBuffer 4?
A6: 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.
