R0133 FAQs for SpeI, Restriction Endonucleases, Intl, NEB
New England Biolabs
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SpeI FAQ

Q1: Are there any common problems encountered when using SpeI?
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?
Q3: Has the conversion to NEBuffer 4 altered any of the properties of the restriction enzyme?
Q4: If I have an old tube of enzyme, what NEBuffer should I use?
Q5: Will the new enzyme work in the originally supplied NEBuffer?
Q6: Why is NEB switching this restriction enzyme to NEBuffer 4?
Q7: Can SpeI be used at alternative reaction temperatures?
Q8: Is SpeI active at 25C?
Q9: Does SpeI produce commonly used compatible ends?

Q1: Are there any common problems encountered when using SpeI?

A1: No.


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.


Q7: Can SpeI be used at alternative reaction temperatures?

A7: Incubation of SpeI at 45C results in an increase in activity.


Q8: Is SpeI active at 25C?

A8: It is only 0-25% active.


Q9: Does SpeI produce commonly used compatible ends?

A9: SpeI cleaves to leave a 5 CTAG which can be ligated to fragments generated by AvrII, NheI, StyI (C/CTAGG), and XbaI.