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Buffers in Molecular Biology
 
  Gomori buffers are the most commonly used phosphate buffers and they consist of a mixture of monobasic dihydrogen
phosphate
and dibasic monohydrogen phosphate. Here is a template to prepare phosphate buffers with different pH and strength.
 
 
   
 
 

pH     Buffer Strength mM

MW_Na2HPO4 D   MW_NaH2PO4 D

Monosodium phosphate, Na2HPO4 g/L

Disodium phosphate, NaH2PO4 g/L

   
 
  Type the desire pH and strength of the buffer. Check the Molecular Weight  (MW) of your Phosphate Compounds  and enter the values. Don't forgate to check the pH by pH meter.  
     
 

Considerations:

Phosphate buffers must be avoided in DNA preps which are going to be used for cloning. A phosphate ion inhibits many enzymatic reactions including cleavage of DNA by many restriction enzymes, ligation of DNA, and bacterial transformation. (Phosphates sequester bivalent cat ions such as Ca2+ and Mg2+.) Once added in DNA solutions phosphate salts are very difficult to be removed by ethanol precipitation because they easily co-precipitate with DNA.  However Phosphate buffers are perfect for protein extraction and storage buffers.

Use phosphate buffer at list at 10mM concentration to as high as you can get (1M evan higher if the phosphate doesn’tprecipitate).

 
  Theory behind:  
 
Henderson-Hasselbalch equation
pH = pKa + log [OH-]/[H]

here

[OH-] base;

[H] acid;

pKa is the -log of the weak acid dissociation constant;

log means the base ten logarithm.

 
 

How do you calculate this thing? In the formula above, pH is the value we are looking for. pK is log value of the dissociation constant. Because it contains three acidic protons, Phosphoric acid has three dissociation constants and each of the three can be used to create buffers for either of the three corresponding pH ranges. The three pKa values for phosphoric acid are 2.15, 6.86 and 12.32. Monosodium phosphate and its conjugate base, disodium phosphate are usually used to generate buffers of pH values around 7, for biological applications, as is shown here.

pK = 2.15= log(NaH2PO4/H3PO4); pK=6.86=log(Na2HPO4/NaH2PO4); pK=12.32= log((Na3PO4/Na2HPO4)

From here it is obvias if you keep the ratio; base/acid=1, pH is the coresponding pK value. To change the pH we simply change the ratio.

Still not sure what is going on, check this link for detail tutorial on this matter.

 
  Table of common buffers in Mol Biology  

Common Name

pKa
at 25°C

Buffer Range

Temp Effect
(pH / °C)**

Mol.
Weight

Full Compound Name

TAPS

8.43

7.7 – 9.1

−0.018

243.3

3-{[tris(hydroxymethyl)methyl]amino}propanesulfonic acid

Bicine

8.35

7.6 – 9.0

−0.018

163.2

N,N-bis(2-hydroxyethyl)glycine

Tris

8.06

7.5 – 9.0

−0.028

121.14

tris(hydroxymethyl)methylamine

Tricine

8.05

7.4 – 8.8

−0.021

179.2

N-tris(hydroxymethyl)methylglycine

HEPES

7.48

6.8 – 8.2

−0.014

238.3

4-2-hydroxyethyl-1-piperazineethanesulfonic acid

TES

7.40

6.8 – 8.2

−0.020

229.20

2-{[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid

MOPS

7.20

6.5 – 7.9

−0.015

209.3

3-(N-morpholino)propanesulfonic acid

PIPES

6.76

6.1 – 7.5

−0.008

302.4

piperazine-N,N′-bis(2-ethanesulfonic acid)

Cacodylate

6.27

5.0 – 7.4

138.0

dimethylarsinic acid

MES

6.15

5.5 – 6.7

−0.011

195.2

2-(N-morpholino)ethanesulfonic acid

NaPhospate
12.4
4.8-8.8
−0.005
169
NaH2P04/Na3PO4

Acetate

4.76

3.8 – 5.8

59.04

Deprotonated Ethanoic Acid (Non-IUPAC Name);

 
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