Luckau Protocols:Tris-Cl

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Tris-Cl Protocol

Tara K. Luckau's Home Page

Conservation Genetics Lab Notebook

Tara's Protocols


Contents

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


  • Note: when adjusting pH, be patient: adjust the pH slowly until it seems steady and stable, then leave the solution on the stirrer for an hour or so, then re-check the pH to be sure it hasn't changed. It's always a good idea to re-re-check the pH a day after as well.


To make \tfrac{1}{2} liter of 0.5M Tris-Cl, pH 8.0

Calculations

  • 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}


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 (remember to be patient: adjust, let it sit, adjust again)
  5. Add H2O to bring volume to 500 mL
  6. Re-check pH
  7. Autoclave solution, store at room temperature


To make \tfrac{1}{2} liter of 1M Tris-Cl, pH 8.0

Calculations

  • 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}


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 (remember to be patient: adjust, let it sit, adjust again)
  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

  • 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}


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 (remember to be patient: adjust, let it sit, adjust again)
  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
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