Additional Exercises in Using Micropipettors

NOTE: This exercise should be preceded by the Learning to Use a Micropipettor activity.

Purpose: To practice the proper use of micropipettors and develop a concept of a microliter (ml).

Micropipetting Activity   part A

Materials: micropipettors: 0.5-10 ml,10-100 ml,100-1000 ml; Distilled water; Methylene blue or other dye stock in 1.5 mL tubes;* Parafilm or wax paper or freezer paper or laminated graph paper.

Procedure: using proper procedure as practiced in the Learning to Use Micropipettors referenced above: You may also want to view the PowerPoint on micropipetting before beginning.

Take the 0.5-10 ml micropipettor, put on appropriate tip and

1. Take a piece of parafilm* and place 5 individual 10 ml drops of water on it.
2. Transfer 5 ml of methylene blue into the first drop. Mix.
3. Transfer 4 ml of methylene blue from stock into the second drop. Mix
4. Transfer 3 ml of methylene blue from stock into the third drop. Mix.
5. Transfer 2 ml of methylene blue from stock into the fourth drop. Mix.
6. Transfer 1 ml of methylene blue from stock into the fifth drop. Mix.
   

10 ml is approximately the size of __________________.

Alternately, take the 10-100 ml micropipettor, put on appropriate tip and

1. Take a piece of parafilm* and place 5 individual 100 ml drops of water on it.
2. Transfer 50 ml of methylene blue into the first drop. Mix.
3. Transfer 40 ml of methylene blue from stock into the second drop. Mix
4. Transfer 30 ml of methylene blue from stock into the third drop. Mix.
5. Transfer 20 ml of methylene blue from stock into the fourth drop. Mix.
6. Transfer 10 ml of methylene blue from stock into the fifth drop. Mix.

Alternately, take the100-1000 ml micropipettor, put on appropriate tip and

1. Take a piece of parafilm* and place 5 individual 1000 ml drops of water on it.
2. Transfer 500 ml of methylene blue into the first drop. Mix.
3. Transfer 400 ml of methylene blue from stock into the second drop. Mix
4. Transfer 300 ml of methylene blue from stock into the third drop. Mix.
5. Transfer 200 ml of methylene blue from stock into the fourth drop. Mix.
6. Transfer 100 ml of methylene blue from stock into the fifth drop. Mix.
   1000 ml is equal to _______________ mL.

Observations: 

1. Create a table and record your results as measure of intensity of color.

2. Compare the size of 600 ml, 900 ml, and 1200 ml drops to common coins:

600 ml is approximately equal in size to:__________________

900 ml is approximately equal in size to:__________________

1200 ml is approximately equal in size to:_________________

3. Alternatively, measure the diameter of above size drops and record in a table.

Set the 1000 ml pipettor to 1000 to "clean up" the drops onyor paper and expel the drops into the water cup. This cup will be used for the Extension of the next page.   POUR UNDILUTED METHYLENE BLUE BACK INTO THE STOCK BOTTLE.

Micropipetting Activity   part B

Sol I  40 ml of distilled water and two drops of red food coloring
Sol II  40 ml of distilled water and two drops of yellow food coloring
Sol III 40 ml of distilled water and two drops of blue food coloring
Sol IV 40 ml of distilled water and one drop of green food coloring

1. Pick up a small-volume Micropipettor (use the 0.5-10 or 1-10 µl pipettor).
2. Label three 1.5 ml microcentrifuge tubes with A, B, and C.

Use the matrix below as a guide to adding solutions to each tube.

TUBE	Sol I	Sol II	Sol III	Sol IV
A	4 µl	5 µl	1 µl	-
B	4 µl	5 µl	-	1 µl
C	4 µl	4 µl	1 µl	1 µl

1.  Set micropipettor to 4 µl and add Solution I to each tube.
2. Use a fresh tip to add appropriate volume of Solution II to a clean spot on reaction tubes A, B, and C.
3. Use a fresh tip to add 1 µl of Solution III to tubes A and C
4. Use a fresh tip to add 1µl of Solution IV to tubes B and C
5. Close tops, pool and mix reagents in tubes by sharply tapping tube bottom on bench top.
6. A total of  _____µl has been added to each tube.  To check your measurements, set the pipettor to 10 µl and very carefully withdraw solution from each tube.
1. Is the tip just filled?
2. Is there even a tiny volume of fluid left in the tube?

1. Pick up a large-volume Micropipettor (use the 100-1000 µl pipettor)
2.  Label two 1.5 ml microcentrifuge tubes E and F

Use the matrix below as a guide to adding Solutions I-IV to these tubes

TUBE	Sol I	Sol II	Sol III	Sol IV
E	100 µl	200 µl	150 µl	550 µl
F	150 µl	250 µl	350 µl	250 µl

1. Set micropipettor to add appropriate volumes of Solutions I-IV to tubes E and F.
2. How many µl of fluid has been added to tubes E and F? _________

1.  Metric Conversions (Liter, L – milliliter, ml – microliter, µl) for liquid measurements.  The two most useful units in molecular biology are the milliliter and microliter.

1 ml = 0.001 liter                            1000 ml = 1 liter
1 µl – 0.000001 liter                       1,000,000 µl = 1 ml

Complete the following conversions:

1 µl =  _________ml                      1 ml = ________µl
10 µl = _________ml                     100 µl = _________ml

Extension I - go to It's In My Genes for a whole class effort using micropipettors and measurement conversions.

Extension II. (addresses measurement objectives as well as micropipetting techniques.)

1. Put droplets of diluted methylene blue of the assigned sizes below on laminated graph paper and measure the diameter of each drop. Record. Graph the volume of the drop against the diameter of the drop.

Volume of Drop                                                           Diameter of drop in mm

10 ml

100 ml

150 ml

1000 ml

1300 ml

1500 ml

2. Measure and weigh 1000 ml of distilled water on a balance.  One mL of water should weigh 1 g.  Repeat 10 times, recording weight 10 times on the table below.

1000 ml drop                                                    Weight of drop in g

Trial 1

2

3

4

5

6

7

8

9

10

Accuracy – is an expression of how close a measurement instrument comes to the true or accepted value – 1.0 mL of water should weigh 1.0 g. Look at your values.  Comment on the accuracy of your micropipettor:

Precision – (repeatability) is an expression of how close in value are repeated measurements on the same instrument.    One simple way of indicating precision is looking at the range of values:  range is the different between the highest and lowest values of a set of measurements.  Using the table above,
Highest value_______   Lowest value: __________  Range: ____________ The more narrow the range, the more precise the instrument. 
Comment on the precision of your micropipettor using Range:

Optional:  A more common way to evaluate the variability of a set of measurements would be to calculate the mean and then the standard deviation.  The lower the standard deviation, the better the precision.  Look up the formulas for calculating mean and standard deviation, perform on your set of repeated measurements for weight versus volume, and re-evaluate the precision of your micropipettor.

Credits: some of these activities were derived from the classic DNA Science by Micklos and Freyer.