Dilution and Pipetting Lab
Dilution and Pipetting Lab
Dilution and Pipetting Lab
Gautam Dhar
Concentration:
Units of concentration:
A. Percent concentration: units of solute dissolved in 100 unit of solution.
% (m/m) = g solute x 100 / g solution
% (m/v) = g solute x 100 / mL solution
% (v/v) = mL solute x 100 / mL solution
B. Molarity: Molarity is defined as the number of moles of solute in 1L of solution
M= moles/L solution
Dilution
In laboratory we often need to dilute solutions from a concentrated stock. If C1 and V1 be the
concentration and volume of the stock and C2 and V2 are the concentration of the diluted solution
respectively then,
C1V1 = C2V2
C1/C2 = V2/V1 = dilution factor (DF)
If a laboratory uses 100 mM NaCl for its experiment but the stock is 1 M then the stock is 10X
concentrated. To make a working solution one has to take 1 vol of the stock and add 9 volume of water.
Or in other word, make the total solution volume 10 times to that of the volume of the stock taken.
Serial Dilution
Often in laboratory we use chemical that are very potent and need to be diluted many times (1000-
100000 fold). In those cases, instead of doing 1 step dilution we do serial dilution. For example, a stock
solution of a chemical is 10000X concentrated. To make a working solution we need to dilute it 10000
times. If we start with 1 mL of stock we need to prepare a 10L solution which is rather a wastage.
Instead. We can perform a serial dilution as shown below. In each step we will dilute the solution 10
times
1. Take 1 mL stock and water to a total volume of 10 mL. This will generate a 1000X stock
2. Take 1 mL 1000X solution and water to a total volume of 10 mL. This will generate 100X stock.
3. Repeat two more times and you will get a 10 mL 1X stock for your work.
Each of these sub-stocks can be labeled and stored for future use.
In general
Final DF = (D1) x (D2) x (D3)……
D1, D2, D3…. represents the fold dilution done in each step.
Instructor: Dr. Gautam Dhar
Prelab:
1. 1 mL of a stock solution of Coomassie Blue reagent with a concentration of 34 mg/mL is poured into a
test tube. Then 4 mL of H2O is added to the test tube.
2. You performed a 1:25 dilution to a stock solution of Bovine Serum Albumin, BSA (2.5 g/mL).
b. If you poured 1 part stock BSA into a test tube to perform this dilution, how many parts of H2O would
you need to dilute this to its proper concentration?
c. If you poured 2.4 mL of stock BSA into a test tube to perform this dilution, how much H2O would you
need to add to dilute this to its proper concentration?
Instructor: Dr. Gautam Dhar
3. You performed a 4-step serial dilution to a stock solution (2.5 g/L). The sequence of dilution factors
is as follows: 1st (2X), 2nd (10X), 3rd (5X), 4th (150X)
a. What is the final concentration of the solution after the 3rd serial dilution?
b. On the 4th step serial dilution, you perform a 150X. If your final volume is 3 mL after the dilution, what
is your initial volume (in L) would you need of your previous dilution to correctly perform this last step?
c. What is the overall dilution factor of your serial dilution from steps 1 through 4?
4. You are given a test tube containing 10 mL of a solution with 8.4 x 107 cells/mL. You are to produce a
solution that contains 84 cells/mL. You will perform a 3-step serial dilution in your laboratory.
Draw test tubes and properly label concentrations after each step of your serial dilution. Also indicate the
dilution factors for each step along with the volumes of solution and H2O needed to dilute each step.
Instructor: Dr. Gautam Dhar
Procedure:
Data Sheet:
Part 1:
A. 2-20 ul pipette
Volume (µl) Tial 1 Trial 2 Trial 3 Average SD
Unit:-
2
10
15
20
Equation:
R2 =
B. 20-200 ul pipette
Volume (µl) Tial 1 Trial 2 Trial 3 Average SD
Unit:-
25
50
75
100
150
200
Equation:
R2 =
Instructor: Dr. Gautam Dhar
C. 200-1000 ul pipette
Volume (µl) Tial 1 Trial 2 Trial 3 Average SD
Unit:-
250
500
750
1000
Equation:
R2 =
Data Table 1. Determining Mass Percent & Average Densities of Known Sugar Solutions
Mass of Mass of Mass Mass of 1.000 mL Average Average
Sugar Solution Percent of Solution (g) Mass of Density of
Solution
(g) (g) Sugar Determination Solution Solution
(%) 1 2 3 (g) (g/mL)
A
E 0.000 g 0.00%
Graph:
1. Using Microsoft excel or similar program, input the data from Mass Percent Sugar (%) and Average
Density of Solution (g/mL). Input the data so that the Mass % Sugar is the independent variable (x-axis)
and the Average Density of each solution as the dependent variable (y-axis).
2. Be sure to label the axes and title the graph appropriately.
3. Perform a linear regression analysis. Include the equation of the trend line and correlation value.
1. What information do you get from the slope of the line in part A for each graph? Is it same for all the
curve? If so, why?
2. What are the dilution factors of D1, D2, D3, D4 and D5 in Part2 table 2?