Question

Record below how you would make 20 ml of this reagent, using the stock solutions indicated 1 M Tris-HCI (pH 6.8): 100 glycerol: 10% SDS: bromophenol blue (solid): H20: (to 90% of final volume to allow for later addition of β-mercaptoethanol)

Answer 1

Answer:

1M Tris-HCL (pH 6.8): 2.6mL

100% glycerol:4mL

10% SDS:8mL

Bromophenol blue (solid): 20mg

H2O: 7.4mL

NOTE: required 10% (vol/vol) B mercaptoethanol.

Explanation:

[2X SDS sample buffer: 130 mM Tris-HCl, pH 6.8 20% (vol/vol) glycerol 4% (wt/vol) SDS I mg/ml bromophenol blue 10% (vol/vol) β-mercaptoethanol (add just before use) Given data, 1 M Tris-HCI (pH 6.8) 100 glycerol 10% SDS STEP 1: Calculation of 90% of total volume 20mL ? 20mL of reagent need to make. Of which 10% (vol/vol) B-mercaptoethanol So, remaining 90% volume all other components 90% volume= (90 X 20)/100 = 18mL STEP2: Calculation of required volume of 1M Tris-HCI stock to get final concentration of 130mM of Tris-HCI, pH 6.8 Note: 1M of Tris-HCl-1000mM of Tris-HCI Required volume in mL= ( 20mLX1 30mM)/1 000mM = 2.6mL STEP3: Calculation of required volume of 100% (vol/vol) glycerol Final concentration is 20% (means out of 100mL solution 20mL of glycerol). Therefore, in 20mL reagent to get 20% using 100% glycerol is F(20 X20)/1004 mL STEP4: Calculation of required volume of 10% SDS to get 4% final concetration in 20mL. =(4% X 20)/10%-8mL STEP5: Calculation of required amount of bromophenol blue Final conc. is 1 ma/mL Therefore, for 20mL = 20 X 1mg-20mg/20mL STEP6: Calculation of required volume of water to get Final 90% volume (18mL) Therefore, required volume of water. Final 90% volume-all the componets volume 18mL-(2.6mL TrisHCl +4mL100% glycerol + 8mL of 10% SDS) 18mL-10.6mL =7.4mL of water STEP7: Calculation of 10% (vol/vol) B-mercaptoethanol Total volume- final volume of all the components in the above calculation 20-18mL -2mL