Question

(A) Your whole cell lysate sample gives an absorbance reading of 15 when undiluted. What dilution would be necessary to get the absorbance down to 0.5?

(B) You want a total whole cell lysate sample volume of 1.0 mL to retest its absorbance. Using the dilution factor you determine in part (A), what volume of the whole cell lysate sample and what volume of buffer would you use to make the dilution?

Answer 1

(A) The undiluted sample gives an absorbance of 15 AU (AU = absorbance unit) while the diluted sample gives an absorbance of 0.5 AU.

The dilution factor (df) is given by (Absorbance of undiluted sample)/(Absorbance of diluted sample) = (15 AU)/(0.5 AU) = 30 (ans).

(B) The total volume of the diluted sample is 1.0 mL; therefore, the volume of the undiluted sample required = (total sample volume)/(df) = (1.0 mL)/(30) = 0.0333 mL = (0.0333 mL)*(10³µL/1 mL) = 33.3 µL (ans).

The volume of buffer required to make up the volume of the sample = (1.0 – 0.333) mL = 0.9667 mL = (0.9667 mL)*(10³ µL/1 mL) = 966.7 µL (ans).