Luckau Protocols:Tris-Cl
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Tris-Cl Protocol |
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
- Using digital scale, place 39.39 g Tris-base powder into weight boat
- In 1 L beaker, mix Tris-base powder in 400 mL H2O
- Put beaker on hotplate with stirbar until dissolved
- 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)
- Add H2O to bring volume to 500 mL
- Re-check pH
- 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
- Using digital scale, place 78.78 g Tris-base powder into weight boat
- In 1 L beaker, mix Tris-base powder in 400 mL H2O
- Put beaker on hotplate with stirbar until dissolved
- 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)
- Add H2O to bring volume to 500 mL
- Re-check pH
- 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
- Using digital scale, place 30.285 g Tris-base powder into weight boat
- In 250 mL glass media bottle, mix Tris-base powder in approximately 200 mL H2O
- Put beaker on stirrer with stirbar until dissolved
- 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)
- Add H2O to bring volume to 250 mL
- Re-check pH; the next day, re-check it again
- Autoclave solution, store at room temperature