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Buffers

HEPES

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  • 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
  • Zwitterionic sulfonic acid buffering agent
  • White crystalline powder
  • Maintains physiological pH in cells
  • Must be kept in darkness in the presence of riboflavin to prevent H2O2 formation
  • Useful pH range: 2.5-3.5 or 6.8-8.2
  • Doesn't bind metal ions, thus a good buffer for enzymes that may be inhibited by metal chelation
  • When pH is adjusted with KOH, it is called HEPES-K

Tris(hydroxymethyl)aminomethane (Tris)

Overview

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  • Base
  • Component in buffers, such as TAE and TBE
  • Condensates with aldehydes
  • Complexes with metal ions
  • Effective pH range: 7.1-9.1
  • Inhibits many enzymes either directly or via chelation

Preparation

To make the solution of a required pH, use Henderson-Hasselbach equation:

\[pH = pK_a + \log_{10} \frac{[\text{Tris base}]}{[\text{Tris-HCl}]}\]

At 25C, pKa = 8.1 (sources vary on the precise value). If the required pH is 8.0, then

\[d = \frac{[\text{Tris base}]}{[\text{Tris-HCl}]} = 10^{pH-pK_a} = 10^{-.1} = .794\]

Since

\[[\text{Tris base}] + [\text{Tris-HCl}] = [\text{Tris buffer}]\]

we can compute Tris base concentration:

\[[\text{Tris base}] = \frac{d}{d + 1} [\text{Tris buffer}] = .460\]
\[[\text{Tris-HCl}] = .540\]

To make 10 mL of 1 M Tris-HCl:

MW (g/mol) Final conc. (M) Final vol. (mL) Amount to weigh (mg)
Tris base 121.136 .44 10 533
Tris-HCl 157.60 .56 10 882.56

However, in practice the expected pH is not necessarily observed and needs to be adjusted with HCl or NaOH.

Alternative

This method avoids using Tris-HCl but might require a huge amount of HCl. For 1M solution: 1. Trizma (or Tris) base: 12.1 g in 60 mL water 2. HCl (concentrated): add a drop at a time until the desired pH is reached 3. Fill up to 100 mL

Resources

Tris/EDTA (TE)

  • Solubilizes DNA and RNA and protects from degradation
  • pH 8
  • 10 mM Tris-HCl
  • 1 mM EDTA

Tris/Acetate/EDTA (TAE)

  • Tris-acetate buffer provides a pH of 8.3
  • EDTA binds metal ions
  • Used for dsDNA and RNA separation in electrophoresis
  • Linear dsDNA runs faster than TBE
  • But lower buffer capacity than TBE
50X TAE components Amount
Tris base (g) 242
Glacial acetic acid (mL) 57.1
.5 M EDTA  (pH 8.0; mL) 100
ddH20 (mL) to 1 L

Tris/Borate/EDTA (TBE)

  • Similar to TAE, but higher buffer capacity
10X TBE components Amount
Tris base (g) 108
Boric acid (mL) 55
.5M EDTA  (pH 8.0; mL) 40
ddH20 (mL) to 1 L

Sodium acetate

  • Buffer in mildly acetic range (pH 4-6)

10X NucleaSeq

Source: Massively parallel kinetic profiling of natural and engineered CRISPR nucleases | Nature Biotechnology

  1. Prepare 980 uL of all ingredients except DTT that is not stable in solution and should be added each time from stock aliquots or fresh preparation.
Component Input vol. (uL) Stock conc. (M) Final conc. (M)
HEPES (pH 7.5) 200 1 200
KCl 500 3 1500
MgCl2 100 1 100
Water 180
Total 980
  1. Check that the mix has a pH around 7.5. If not, adjust with KOH / HCl.
  2. Prepare 1 M DTT:
    1. 15.4253 mg DTT (this is a lot of DTT as compared to the amount of water, but it easily dissolves)
    2. 100 uL water
  3. To make 50 uL of the 10X buffer with 20 mM DTT final concentration:
    1. 49 uL buffer
    2. 1 uL DTT
  4. Split into aliquots of 50 uL and store at -20C.