R0146 FAQs for XhoI, Restriction Endonucleases, Intl, NEB
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XhoI FAQ

Q1: Why isn't XhoI cutting?
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: Does XhoI produce commonly used compatible ends?
Q8: How does XhoI differ from its isoschizomer, PaeR71?
Q9: What is the molecular weight of XhoI?
Q10: What is the activity of XhoI at 25C?

Q1: Why isn't XhoI cutting?

A1: 1. Blocked by CpG methylation.
2. Supercoiled plasmids may require up to 10-fold more XhoI for complete digestoin than linear DNA.


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: Does XhoI produce commonly used compatible ends?

A7: XhoI cleaves to leave a 5 overhang which can be ligated to fragments generated by SalI and AvaI digestion.


Q8: How does XhoI differ from its isoschizomer, PaeR71?

A8: The cleavage sequence of XhoI is identical to that of PaeR71. However, it has been determinded experimentally that the sequence ctCTCGAG is resistant to cleavage by PaeR71. XhoI can be heat inactivated while PaeR71 can not. Both are blocked by CG methylation.


Q9: What is the molecular weight of XhoI?

A9: The molecular weight of XhoI is 26kDa based on PAGE estimation.


Q10: What is the activity of XhoI at 25C?

A10: XhoI is 50-75% active at 25C.