Buffers in Molecular Biology |
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| 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. |
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| 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). |
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| Theory behind: | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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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. |
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| Table of common buffers in Mol Biology | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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