Presentation on theme: "PREPARING LABORATORY SOLUTIONS AND REAGENTS II."— Presentation transcript:
http://matcmadison.edu/biotech/ PREPARING LABORATORY SOLUTIONS AND REAGENTS II
Solutions With More Than One Solute RECIPE I Na 2 HPO 4 6 g KH 2 PO 4 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
http://matcmadison.edu/biotech/ RECIPE II 1 M MgCl 2 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 With More Than One Solute Cont…
http://matcmadison.edu/biotech/ A Recipe With Multiple Solutes, SM Buffer SM BUFFER 0.1 M NaCl 1 mM MgSO 4 0.2 M Tris, pH 7.5 0.01% gelatin This might be whole recipe as given in a manual
http://matcmadison.edu/biotech/ 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.
http://matcmadison.edu/biotech/ 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.
http://matcmadison.edu/biotech/ 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 MgSO 4 requires 1/1000 of its MW. MgSO 4 comes in more than one hydrated form; read container to determine MW. Weigh out correct amount. Add it to Tris buffer.
http://matcmadison.edu/biotech/ 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.
http://matcmadison.edu/biotech/ Strategy 2: PREPARING SM BUFFER WITH STOCK SOLUTIONS In overview: Four solutes are each prepared separately as concentrated stock solutions.
http://matcmadison.edu/biotech/ When the four stocks are combined, they dilute one another to the proper final concentrations
http://matcmadison.edu/biotech/ 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.
http://matcmadison.edu/biotech/ 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 MgSO 4 in water, bring to a volume of 100 mL.
http://matcmadison.edu/biotech/ 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.
http://matcmadison.edu/biotech/ 4.Prepare a stock solution of gelatin, for example, 1%. a. Dissolve 1 g in a final volume of 100 mL of water.
http://matcmadison.edu/biotech/ 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 C 1 V 1 = C 2 V 2 equation four times, once for each solute.
http://matcmadison.edu/biotech/ For example, to calculate how much Tris stock is required: C 1 V 1 = C 2 V 2 (1 M) (?) (0.2 M) (1000 mL) ? = 200 mL
http://matcmadison.edu/biotech/ Summary To make 1000 mL of SM buffer, combine: 200 mL Tris stock 1 mL MgSO 4 stock 100 mLNaCl stock 10 mL Gelatin stock BTV 1L; check and record pH.
http://matcmadison.edu/biotech/ 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.