WENDY CHAO'S LABORATORY PROTOCOLS

RECIPES FOR COMMON SOLUTIONS AND BUFFERS

0.5 M EDTA, pH 8.0

• 186.1 g EDTA disodium salt (Na₂EDTA-2H₂O)
• 700 mL dH₂O
• ~50 mL 10N NaOH to pH 8.0

  Stir until dissolved; bring volume to 1 L with dH₂O. Filter and autoclave if desired (but really, nothing will grow in this). Store at room temperature.

10N NaOH (sodium hydroxide)

• Dissolve 400 g sodium hydroxide pellets in 800 ml dH₂O (the solution will heat up quickly)
• Cool; bring volume to 1 L
• Store at room temperature in plastic bottles (will erode glass)

TBE (Tris-borate-EDTA), 10X stock*

• 108 g tris base (TRIZMA)
• 55 g boric acid
• 40 to 50 mL 0.5 M EDTA (pH 8.0) (or 7.4 to 9.3 g EDTA)**

  Add the above to 800 mL dH₂O; stir until dissolved. Adjust to 1 L* with additional dH₂O. Store at room temperature.

  *Some protocols recommend making TBE stock at 5x instead of 10x to prevent boric acid precipitation. To make 5x TBE, simply adjust the final volume to 2 L instead of 1 L. However, 5x takes up twice as much real estate in a crowded lab as 10x stock; furthermore, the boric acid will eventually precipitate out of 1x TBE anyway. I recommend just sticking with 10x TBE stock - if the borate concentration is slightly off, your gels won't suffer. This ain't analytical chemistry. You could also try adding a few drops of 10N NaOH. The borate will go into solution like magic. Actually, it's not magic, it's chemistry—just not analytical chemistry.

  **The amount of EDTA in TBE may vary by protocol. Again, this ain't analytical chemistry.

1 M Tris-HCl

• Dissolve 121.1 g Tris Base (TRIZMA) in 700 ml dH₂O
• Add concentrated HCl to desired pH:
  • pH 7.4: 70 mL
  • pH 8.0: 42 mL
  • pH 9.0: ~8 mL
• Bring volume to 1 L with dH₂O. Filter and autoclave if desired.

  Store at room temperature.

TE Buffer (10 mM Tris, 1 mM EDTA)

• 0.5 mL 1 M Tris-Cl, pH 7.4 (sterile)
• 0.1 mL 0.5 M EDTA, pH 8.0 (sterile)
• 49.4 mL ddH₂O (sterile) to 50 mL. Store at room temperature.

10X Loading Buffer - 25% Ficoll, 100 mM Tris-HCl & EDTA, Xylene Cyanol, Orange G

• 2.5 g Ficoll-400
• 1 mL 1 M Tris-Cl, pH 7.4
• 2 mL 0.5 M EDTA
• Bring to 10 mL with ddH₂O, heating to 65°C to dissolve.
• Add 25-50 mg each xylene cyanol and orange G.

  If needed, adjust dye intensity with colorless 10x buffer. Store at room temperature.

More about loading buffer and why this is the best loading buffer recipe.

NOTE: I recommend first making 50 mL 10X loading buffer WITHOUT any marker dyes. You can then add different combinations of dyes and dilute them as you wish to attain your preferred color intensity. This will yield enough loading buffer for your entire career, and the careers of your future postdocs and your postdocs' postdocs. You'll also have enough to experiment wildly with various dye combinations!

10X Loading Buffer (25% Ficoll, 100 mM Tris-HCl, 100 mM EDTA, no dye)

• 12.5 g Ficoll-400
• 5 mL 1 M Tris-Cl, pH 7.4
• 10 mL 0.5 M EDTA
• Bring to 50 mL with ddH₂O, heating to 65°C to dissolve.
• To 10 mL of this, add 25-50 mg of various dyes. If needed, adjust dye intensity with colorless 10x buffer. Store at room temperature.

  More about loading buffer and why my particular recipe is superior.