http://matcmadison.edu/biotech/ preparing laboratory solutions and reagents ii
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PREPARING LABORATORYSOLUTIONS AND REAGENTS II
Solutions WithMore Than One Solute
RECIPE I
• Na2HPO4 6 g• KH2PO4 3 g • 0.4% glycerol 10 mL• Dissolve in water. • Bring to a volume of 1 liter.• Recipe lists amounts of solutes.• This is easy to follow
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RECIPE II
• 1 M MgCl2
• 0.1 M Tris• Gives the final concentration of each solute.• You must calculate the amount of each to use. • This is harder to interpret
Solutions WithMore Than One Solute Cont…
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A Recipe WithMultiple Solutes, SM Buffer
SM BUFFER
0.1 M NaCl
1 mM MgSO4
0.2 M Tris, pH 7.50.01% gelatin
• This might be whole recipe as given in a manual
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Strategy 1:
PREPARING SM BUFFER WITHOUT STOCK SOLUTIONS, OVERVIEW:
• Prepare a solution of 0.2 M Tris, pH 7.5. • Calculate grams of each of the other solutes
required. • Weigh out solutes and dissolve directly in Tris
buffer.
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Strategy 1
• Decide how much buffer to make, for example, 1 liter.
• 1 liter of 0.2 M Tris requires 24.2 g of Tris base. (MW = 121.1 g/mole.)
• Dissolve Tris in about 700 mL of water and bring pH to 7.5.
• Do not bring Tris to volume.
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• 4. 1 liter of 0.1 M NaCl requires 5.84 g of NaCl. Add this to Tris buffer.
• 5. 1 liter of 1 mM MgSO4 requires 1/1000 of its MW.
• MgSO4 comes in more than one hydrated form; read
• container to determine MW.• Weigh out correct amount.• Add it to Tris buffer.
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• 0.01% gelatin is 0.1 g in 1 L. Weigh out and add to Tris buffer.
• Stir to dissolve• BTV 1 L• Check and record pH.
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Strategy 2:
PREPARING SM BUFFER WITH STOCK SOLUTIONS
• In overview: • Four solutes are each prepared separately as
concentrated stock solutions.
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• When the four stocks are combined, they dilute one another to the proper final concentrations
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Steps Are:
1. Prepare a stock solution of Tris buffer at pH 7.5.
a. No set rule as to what should be concentration of stock.
b. Example: To make 1 L of 1 M stock, dissolve 121.1 g of Tris base in about 900 mL of water.
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c. Bring pH to 7.5.
d. BTV 1L.
2. Prepare a stock solution of magnesium sulfate, for example, 1 M.
a. To make 100 mL, dissolve 0.1 MW of MgSO4 in water, bring to a volume of 100 mL.
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3. Prepare a stock solution of NaCl, for example,1 M.
a. To make 100 mL of stock, dissolve 5.84 grams in water and BTV 100 mL.
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4. Prepare a stock solution of gelatin, for example, 1%.
a. Dissolve 1 g in a final volume of 100 mL of water.
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5. To make the final solution, combine the right amounts of each stock.
a. Since this is a situation where stocks are diluted, use the C1V1 = C2V2 equation
four times, once for each solute.
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For example, to calculate how muchTris stock is required:
C1 V1 = C2 V2
(1 M) (?) (0.2 M) (1000 mL)
? = 200 mL
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Summary
To make 1000 mL of SM buffer, combine:
200 mL Tris stock
1 mL MgSO4 stock 100 mL NaCl stock 10 mL Gelatin stock
BTV 1L; check and record pH.
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Which Strategy?
• Both strategy 1 and 2 are correct. • Generally efficient to make stock solutions of
frequently used solutes because weighing out chemicals is time-consuming.