Thermo packages a unique buffer with EcoRI:

  • 50 mM Tris-HCl (pH 7.5)
  • 10 mM MgCl2
  • 100 mM NaCl
  • 0.02% Triton X-100
  • 0.1 mg/mL BSA

However, they also list their EcoRI enzyme as 100% active in Buffer O with no indicated star activity:

  • 50 mM Tris-HCl (pH 7.5 at 37°C)
  • 10 mM MgCl2
  • 100 mM NaCl
  • 0.1 mg/ml BSA

The same is true of 2X Tango:

  • 66 mM Tris-acetate (pH 7.9 at 37°C)
  • 20 mM magnesium acetate
  • 132 mM potassium acetate
  • 0.2 mg/ml BSA

Is there a reason one would preferably use the unique buffer over a universal one? Given that buffer compatibility is a major selling point for restriction enzymes, why would they continue to supply a unique buffer?

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    $\begingroup$ I look forward to the explanation for why this is a homework question... $\endgroup$ – canadianer Apr 17 '17 at 19:08
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    $\begingroup$ Me too. I think this is a legitimate question. And an interesting one, too. $\endgroup$ – Chris Apr 17 '17 at 19:28
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    $\begingroup$ Interestingly for the Fast Digest system an universal buffer (green) is recommended. $\endgroup$ – Chris Apr 17 '17 at 19:31
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    $\begingroup$ I have a suspicion that the unique buffer might be technically the best but the enzyme is also almost fully active in the universal buffers and displays neglible star activity so they just list it as 100% active for simplicity's sake. I may write their technical support a short email to see if they can clarify. $\endgroup$ – canadianer Apr 17 '17 at 20:09
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    $\begingroup$ If you do, please let us know when you get an answer that tells us anything (although I doubt it). $\endgroup$ – Chris Apr 17 '17 at 20:13

Activity toward the desired substrate sequence is not the only concern in evaluating a restriction enzyme buffer system. In addition, you need to be concerned about so-called "star activity", or activity of the enzyme toward other, non-desired nucleotide sequences.

For example, NEB supplies an enzyme-specific buffer for it's standard EcoRI, even though it lists 100% activity in buffer #2 of NEB's standard buffer series. But at the very bottom of the page, there's this note: "May exhibit star activity in NEBuffer 2.1 or CutSmart Buffer."

Promega has a similar warning about it's "MULTI-CORE" buffer - even though EcoRI has 100% activity in that buffer. The MULTI-CORE buffer is not recommended due to star activity - cleavage at non-standard sites.

It's a little hidden on the product data sheet, but Thermo Scientific does have a mention of star activity in the standard "Tango" buffer, and mentions star activity with overdigestion in the standard "R" buffer. There's no warning about buffer "O", but given EcoRI's propensity toward star activity, I'm guessing this is part of why they recommend a unique buffer for routine usage. Given how frequently EcoRI is used in molecular biology, it's worth using a custom buffer to get the star activity as low as possible.

From canadianer: I contacted Thermo's technical support. Despite the fact that star activity is not listed as a concern for either 2X Tango or Buffer O in the product data for EcoRI, they say that it may actually show some star activity in these universal buffers which is why they continue to supply the unique buffer for this enzyme.

  • $\begingroup$ Thanks for your answer. I should've clarified in my question that Thermo does not indicate star activity for both Buffer O and 2X Tango. As I mentioned in the comments, I suspect you may be right that star activity is minimized in the unique buffer whereas there may be some higher degree (negligible?) in the universal buffers. $\endgroup$ – canadianer Apr 17 '17 at 23:14

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