Luckau Protocols:Tris-Cl: Difference between revisions

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==Protocol==
==Protocol==
0.5M Tris-Cl, pH 8.0
===0.5M Tris-Cl, pH 8.0===


<math>500 mL \bullet \frac{0.5 mol}{L}\ \bullet \frac{L}{1000 mL}\ \bullet \frac{157.56 g}{mol}\ = 39.39 g</math>
<math>500 mL \bullet \frac{0.5 mol}{L}\ \bullet \frac{L}{1000 mL}\ \bullet \frac{157.56 g}{mol}\ = 39.39 g</math>


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


1M Tris-Cl, pH 8.0
# In 1 L beaker, mix 39.39 g Tris-Cl in 400 mL H<sub>2</sub>O
# Put beaker on hotplate with stirbar until dissolved
# Using concentrated aqueous HCl to adjust to pH 8.0
# Add H<sub>2</sub>O to bring volume to 500 mL
# Autoclave solution, store at room temperature


78.78 grams in 500 mL of distilled water = 1M solution
===1M Tris-Cl, pH 8.0===
Dissolve 39.39 g Tris-Cl in 400 mL
Stir, add heat if not dissolving
adjust to pH 8.0
add DI to 500mL
Atoclave


<math>500 mL \bullet \frac{1 mol}{L}\ \bullet \frac{L}{1000 mL}\ \bullet \frac{157.56 g}{mol}\ = 78.78 g</math>


{| {{table}} style="text-align:center"
'''to make \tfrac{1}{2} liter of 1M Tris-Cl, pH 8.0:'''
|+ '''to make 1 liter of 1x Low TE:'''
|-
! Volume !! Reagent !! [Final]
|-
|20 ml || 0.5M Tris-HCl, pH 8.0 || 10 mM
|-
|0.2 ml  || 0.5M EDTA, pH 8.0 || 0.1 mM
|-
|979.8 ml || ddH<sub>2</sub>O ||
|}


 
# In 1 L beaker, mix 78.78 g Tris-Cl in 400 mL H<sub>2</sub>O
*Sterilize by autoclaving for 20 min at 15 psi (1.05 kg/cm2) on liquid cycle
# Put beaker on hotplate with stirbar until dissolved
*Store at room temperature
# Using concentrated aqueous HCl to adjust to pH 8.0
 
# Add H<sub>2</sub>O to bring volume to 500 mL
==FYI==
# Autoclave solution, store at room temperature
* Note, downstream reactions (PCR) typically require Mg<sup>2+</sup>, potentially making the presence of EDTA in the reaction problematic. So, when using DNA or RNA that was suspended in TE, keep track of the amount of EDTA in the mix to make sure there is still enough Mg<sup>2+</sup> for subsequent reactions to proceed successfully. Each EDTA molecule chelates one Mg<sup>2+</sup> ion.
*Many protocols use TE (10mM Tris-Cl and 1mM EDTA), so note that Low TE is different from TE.
*Some people use TE buffers with different pH's for different applications
**DNA is stored at pH 8 to reduce depurination, which is acid catalyzed
**RNA is stored at a slightly lower pH (7.5) because degradation of RNA is base-catalyzed
**Most downstream reactions will not be influenced by the slightly different pH storage conditions.
 
==Sources==
*OpenWetWare's Materials [[TE]] page
*[http://cshprotocols.cshlp.org/cgi/content/full/2006/3/pdb.rec8018 Cold Spring Harb Protoc; 2006; doi:10.1101/pdb.rec8018]
*[http://www.cytographica.com/lab/solutions/TE.htm Cytographica]

Revision as of 13:31, 22 November 2010

Back to User:Tara_K._Luckau

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Purpose

  • Tris-Cl is a buffering agent (made from Tris base and HCl)
  • We use it as a stock solution for Low TE

Info

  • tris [hydroxymethyl] amino methane (C4H11NO3, 2-Amino-2-hydroxymethyl-propane-1,3-diol)
  • MW = 157.56 g/mol (MW = molecular weight, same as formula weight, FW)

bring to desired pH with concentrated HClaq

Protocol

0.5M Tris-Cl, pH 8.0

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

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

  1. In 1 L beaker, mix 39.39 g Tris-Cl in 400 mL H2O
  2. Put beaker on hotplate with stirbar until dissolved
  3. Using concentrated aqueous HCl to adjust to pH 8.0
  4. Add H2O to bring volume to 500 mL
  5. Autoclave solution, store at room temperature

1M Tris-Cl, pH 8.0

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

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

  1. In 1 L beaker, mix 78.78 g Tris-Cl in 400 mL H2O
  2. Put beaker on hotplate with stirbar until dissolved
  3. Using concentrated aqueous HCl to adjust to pH 8.0
  4. Add H2O to bring volume to 500 mL
  5. Autoclave solution, store at room temperature
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