Luckau Protocols:Tris-Cl

Tris-Cl Protocol

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Purpose

• Tris-Cl is a buffering agent (made from Tris base and HCl)
• In the Clark Lab, 0.5M Tris-Cl, pH 8.0 is used as a stock solution for Low TE
• In the Clark Lab, 1M Tris-Cl, pH 8.0 is used as a stock solution for Low TE
• In the Clark Lab, 1M Tris-Cl, pH 8.3 is used as a stock solution for making the PCR Buffer series (A through H)

Info

• tris [hydroxymethyl] amino methane (C₄H₁₁NO₃, 2-Amino-2-hydroxymethyl-propane-1,3-diol)
  • we currently use Amresco, product code 0497, Lot 2901C127
• MW = 157.56 g/mol

To make [math]\displaystyle{ \tfrac{1}{2} }[/math] liter of 0.5M Tris-Cl, pH 8.0

Calculations

• [math]\displaystyle{ 500 \mathrm{mL} \bullet \tfrac{0.5 \mathrm{mol}}{\mathrm{L}}\ \bullet \tfrac{\mathrm{L}}{1000 \mathrm{mL}}\ \bullet \tfrac{157.56 \mathrm{g}}{\mathrm{mol}}\ = 39.39 \mathrm{g} }[/math]

Protocol

1. Using digital scale, place 39.39 g Tris-base powder into weight boat
2. In 1 L beaker, mix Tris-base powder in 400 mL H₂O
3. Put beaker on hotplate with stirbar until dissolved
4. Use concentrated aqueous HCl to adjust to pH 8.0 (note: to make sure pH is accurate, be patient: adjust the pH slowly until it seems steady and stable, then leave the solution on the stirrer for an hour, then re-check the pH to make sure it hasn't changed)
5. Add H₂O to bring volume to 500 mL
6. Re-check pH
7. Autoclave solution, store at room temperature

To make [math]\displaystyle{ \tfrac{1}{2} }[/math] liter of 1M Tris-Cl, pH 8.0

Calculations

• [math]\displaystyle{ 500 \mathrm{mL} \bullet \tfrac{1 \mathrm{mol}}{\mathrm{L}}\ \bullet \tfrac{\mathrm{L}}{1000 \mathrm{mL}}\ \bullet \tfrac{157.56 \mathrm{g}}{\mathrm{mol}}\ = 78.78 \mathrm{g} }[/math]

Protocol

1. Using digital scale, place 78.78 g Tris-base powder into weight boat
2. In 1 L beaker, mix Tris-base powder in 400 mL H₂O
3. Put beaker on hotplate with stirbar until dissolved
4. Use concentrated aqueous HCl to adjust to pH 8.0 (note: to make sure pH is accurate, be patient: adjust the pH slowly until it seems steady and stable, then leave the solution on the stirrer for an hour, then re-check the pH to make sure it hasn't changed)
5. Add H₂O to bring volume to 500 mL
6. Re-check pH
7. Autoclave solution, store at room temperature

To make 250 mL of 1M Tris-Cl, pH 8.3

Calculations

• [math]\displaystyle{ 250 \mathrm{mL} \bullet \tfrac{1 \mathrm{mol}}{\mathrm{L}}\ \bullet \tfrac{\mathrm{L}}{1000 \mathrm{mL}}\ \bullet \tfrac{121.14 \mathrm{g}}{\mathrm{mol}}\ = 30.285 \mathrm{g} }[/math]

Protocol

1. Using digital scale, place 30.285 g Tris-base powder into weight boat
2. In 250 mL glass media bottle, mix Tris-base powder in approximately 200 mL H₂O
3. Put beaker on stirrer with stirbar until dissolved
4. Use concentrated aqueous HCl to adjust to pH 8.3 (note: to make sure pH is accurate, be patient: adjust the pH slowly until it seems steady and stable, then leave the solution on the stirrer for an hour, then re-check the pH to make sure it hasn't changed)
5. Add H₂O to bring volume to 250 mL
6. Re-check pH; the next day, re-check it again
7. Autoclave solution, store at room temperature