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General Protocol for Performing Serial Dilutions
Overview
When a large dilution is required, an accurate dilution cannot be performed in a single dilution step and, serial dilutions are necessary. The difference between the concentration of the original solution and the concentration of the desired solution (the dilution factor) will determine how many dilutions are required. The most common examples deal with concentration of cells or organisms, or the concentration of a solute. The approximate concentration should be known at the start of the experiment before the appropriate number and amount of dilutions can be made.
Procedure
A. General Concepts of Serial Dilutions
The technique involves the removal of a small amount of an original solution to another container that is then brought up to a predetermined volume using the working solution (i.e. ddH2O, buffer or media).
To make a 1:100 dilution (10-2), remove 10 μl and place this volume in a tube containing 990 μl of ddH2O or media (see Hint #1).
For example, if the original solution contained 5 X 108 cells/ml or contained 2 mg/ml DNA, the concentration has been diluted 100 fold to 5 X 106 cells/ml or 20 μg/ml DNA (see Hint #2). This is often represented as 1:100 or 10-2.
To dilute this by a factor of 1:1000, remove 1 μl of the 1:100 dilution and place it in a tube containing 999 μl of ddH2O or media. The secondary concentration (1:100) has been diluted by a factor of 1,000 and the original solution has been diluted by a factor of 100,000 (the dilution factor).
If the 1:100 solution had a concentration of 5 X 106 cells/ml or 20 μg/ml DNA, we now have a concentration of 5 X 103 (5,000) cells/ml or 20 ng/ml (0.02 μg/ml) DNA.
The power of serial dilutions occurs when the original sample concentration is unknown. If each of the serial dilutions are saved and analyzed; the likelihood of the sample concentration analyzed being within the necessary range is increased (see Section C).
B. Examples of Dilution Calculations
1:2 1 μl added to 1 μl; 10 μl added to 10 μl, etc.
1:5 1 μl added to 4 μl; 10 μl added to 40 μl, etc.
1:10 1 μl added to 9 μl; 10 μl added to 90 μl, 100 μl added to 990 μl, etc.
1:100 1 μl added to 99 μl; 10 μl added to 990, 100 μl added to 9900 μl, etc.
1:1000 1 μl added to 999 μl, 10 μl added to 9990 μl, etc.
C. Example Diluting a Sample 5,000 Fold Using Serial Dilution
The following is an example of serial dilutions in which the concentration of the first solution (1:1) is unknown. Instead of following a standard 1:10, 1:100 and 1:1000 paradigm, variable dilution factors are employed to increase the likelihood of the diluted sample being in the range needed for the analysis. Always keep the serial dilutions until the analysis is complete.
The first dilution is 1:2
1. Label the first microcentrifuge tube 1:2 and add 100 μl of the appropriate buffer or ddH2O (see Hint #3).
2. From the original solution remove 100 μl and add this to the microcentrifuge tube labeled 1:2.
3. Vortex the microcentrifuge tube to mix the solutions.
The second dilution is 1:5
4. Label a new microcentrifuge tube 1:10 and add 400 μl of the appropriate buffer or ddH2O.
5. From the 1:2 dilution, remove 100 μl and add this to the microcentrifuge tube labeled 1:10.
6. Vortex the microcentrifuge tube to mix the solutions.
The third dilution is 1:25
7. Label a new microcentrifuge tube 1:250 and add 480 μl of the appropriate buffer or ddH2O.
8. From the 1:10 dilution, remove 20 μl and add this to the microcentrifuge tube labeled 1:250.
9. Vortex the microcentrifuge tube to mix the solutions.
The fourth dilution is 1:4
10. Label a new microcentrifuge tube 1:1000 and add 750 μl of the appropriate buffer or ddH2O.
11. From the 1:250 dilution, remove 250 μl and add this to the microcentrifuge tube labeled 1:1000.
12. Vortex the microcentrifuge tube to mix the solutions.
The fifth dilution is 1:5
13. Label a new microcentrifuge tube 1:5000 and add 400 μl of the appropriate buffer or ddH2O.
11. From the 1:1000 dilution, remove 100 μl and add this to the microcentrifuge tube labeled 1:5000.
12. Vortex the microcentrifuge tube to mix the solutions.
The five diluted solutions (1:2, 1:10, 1:250, 1:1000 and 1:5000) can all be analyzed. Hopefully, one of the solutions will lie within the range of accuracy and the original concentration of the sample calculated. For example, the concentration determined in the 1:1000 solution was 1 μg/ml therefore the concentration of the original solution would be 1 X 1,000 = 1,000 μg/ml or 1 mg/ml.
Solutions
This bioProtocol does not use any solutions
BioReagents and Chemicals
This bioProtocol does not use any reagents
Protocol Hints
1. The 1:100 refers to the original concentration (1) and the diluted concentration (100). Mathematically we achieve a 1:100 dilution because 10 μl of the original solution is now dissolved in 1000 μl (1 ml) or 1000 / 10 = 100.
2. The original concentration is reduced by the dilution factor. Thus a 2 mg/ml (or 2,000 μg/ml) solution is divided by 100 resulting in a diluted concentration of 2,000 / 100 = 20 μg/ml.
3. The appropriate buffer referenced in this step is a buffer that either the original solution is prepared in OR a buffer that the final solution should be in for the subsequent analysis.
