# Moore Lab TBE

### Background

A TBE Saga

Traditional TBE recipes are weight-based, not molarity-based, which reduces the chance for preparation errors, but reinforces a mindless automaton mentality that plagues students and other lab workers. Also, there is no rationale for the amounts provided, so one is left guessing what the tolerances are for the buffering and divalent metal chelation system. This situation is analogous to LB (lysogeny broth), whose recipes range from the original published by Bertani ("Miller"), and lower NaCl versions ("Lennox" and "Luria").

It is common practice to use traditional TBE at 0.5X for routine agarose gel electrophoresis; not only does this formulation conserve buffer, but there is still plenty of buffering and the gels run cooler because there is less electrical current. We addressed the utility of the recipe and rounded the reagent concentrations to memorable values.

Many labs prepare or purchase 10X TBE, which is 890 mM Tris, 890 mM Borate, and 20 mM EDTA. At this high stock concentration, the reagents will crystallize into a hard mineral that is difficult to remove from storage containers and often throw off concentrations when remaking stocks using the same bottle.

5X is more stable, but still has a tendency to crystallize.

1X TBE would then be at respective concentrations of 89 mM of T and B and 2 mM of E, why? More recently, other established protocols figured out that 0.5X would render better gels, and that some of the previous reagent concentrations were built for lab situations which are now outdated. If gels run fine with 0.5X (45.5mM Tris, 45.5mM Borate, 1 mM EDTA) we thought, why not make the most commonly used form ("1 X") equal to something more convenient for daily use? Thus, our lab uses 1X to represent a TBE Mix of 50mM Tris, 50mM Borate, 1mM EDTA. These are rounded up from 45.5 in the original purely for convenience of calculations and do not appear to significantly alter the gels produced.

### Moore Lab - 5X TBE Stock Recipe

(per liter)

250 mM Tris base

250 mM Boric acid

5 mM EDTA (disodium)

- Put about 800 mLs of  diH2O  in a clean beaker with a stir bar;

- dump in the Tris base and stir;

- dump in the EDTA and wait until dissolved (the high pH of the Tris helps the EDTA go in faster);

- then dump in the boric acid and wait until dissolved. Near the end, squirt around the top of the beaker to rinse in any additional reagent.

- Transfer to a cyliner and top off with beaker-rinse to final volume. Transfer to storage bottle (can remove crystals while waiting for reagents to dissolve).


This is a good storage concentration, but obviously the whole point is to have an easily-accessible and effective working stock at 1X. Dilute it down from here to 1X and keep it nearby, in the event someone leaves you high and dry, it won't take too long to dilute it down from 5X.

Don't be lazy: look at the formula weights on the bottles, confirm the CAS numbers if needed, and calculate it. This method should be the norm for all reagent preparations. This way you won't accidentally use Tris-Cl or non-sodium EDTA.

If you are performing electrophoresis that requires the traditional "1X", just use 2X of this version (100mM Tris & Boric Acid concentrations), it buffers a little better and the current is a bit higher, but not enough to write home about.

Be sure to test your running buffer's conductivity in your setup with the gel you are using before running your protocol. Depending on your application and brand, the correct range may vary. In applications which require the higher amperage, you can just use 2X (on this scale) mentioned previously. Just be aware of your needs and your protocol, that way you can avoid just mindlessly making something that doesn't suit your method and having to backpedal.

### Storage & Cleaning

In the higher-concentration stock bottles, build-up of salt-like crystals will form and eventually cause inconsistencies in your gels.

When crystals are visually apparent or at a regular interval (whichever works for your lab), clean the bottle.

These formations do not effectively remove in water, but are easily dissolved in acetic acid. If bottles are used enough, it may be worth keeping a ~10% Acetic Acid container nearby, with which you can rinse and coat the interior of the bottle and threads of the cap. Typically a quick soak and rub-down with a gloved finger will do the trick but it'll depend on the amount of buildup you've accrued in the threads.

If crystals remain, they will eventually act as nucleation sites for later crystal formation, so really try to make sure you get as much as possible.

An ounce of prevention...

### References

1. Sambrook J, Russell SW, editors. Molecular Cloning: A Laboratory Manual. 3. Cold Spring Harbor Laboratory Press; Cold Spring Harbor, NY: 2001.

2. Sanderson, Brian A et al. “Modification of gel architecture and TBE/TAE buffer composition to minimize heating during agarose gel electrophoresis.” Analytical biochemistry vol. 454 (2014): 44-52. doi:10.1016/j.ab.2014.03.003