Micropipetting Lab

Introduction Micropipetting Lab

Many scientific procedures require the measurement of very small liquid volume amounts. Measurement of these small liquid volumes involves the use of a micropipette capable of measuring as little as one microliter (10-6 liter, 0.000001), or one millionth of a liter. Small errors in pipetting can translate into ENORMOUS errors in experiments. This is due to the fact that solutions you are pipetting are at high concentrations. Inaccurate pipetting is a chief contributor to poor laboratory results.

Take the following precautions when micropipetting:
    -Never use the micropipettor without the tip in place; this could ruin the piston.
    -Never rotate the volume adjustor beyond the upper or lower range of the pipette, as stated by the manufacturer.
    -Never invert or lay the micropipettor down with a filled tip; fluid could run back into the piston.
    -Never let the plunger snap back after withdrawing or expelling fluid; this could damage the piston. Instead gently and slowly draw up the liquid into the pipette tip.
    -Never immerse the barrel of the micropipettor in fluid.
    -Never flame the tip of the micropipettor.
    -Never reuse a tip that has been used to measure a different reagent.

There are essentially three sizes of micropipettors based upon volume ranges: 2-20µl, 20-200µl, 200-1000µl.
Typically the same size tips are used for the small and mid-range volumes with larger tips being necessary for the larger sized micropipettor.

Exercise I

Parts of a micropipette:

Sketch a drawing of the micropipette and identify and label the following parts-
a. Plunger button

b. Tip ejector button

c. Volume adjustment dial

d. Digital volume indicator

e. Shaft

f. Attachment point for a disposable tip

Exercise II

Using the Micropipette:

There are two “stops” when ejecting liquids. Depressing to the first stop measures the desired volume. Depressing to the second stop introduces an additional volume of air to blow out any solution remaining in the tip. Notice these friction stops; they can be felt with the thumb by pushing on the plunger. When drawing liquid into the pipette only go to the FIRST stop; going beyond this initial stop will introduce error into your pipetting and your experiments.

Withdraw 10µl of colored solution from container:
    a. Depress the plunger to first stop and hold it in this position. Dip the tip into the solution to be pipetted, and draw fluid into the tip by gradually releasing the plunger. Be sure that the tip remains in the solution while you are releasing the plunger.
    b. Slide the pipette tip out along the inside of the reagent tube to dislodge any excess droplets adhering to the outside of the tip.
    c. Check that there is no air space at the very end of the tip. To avoid future pipetting errors, learn to recognize the approximate levels to which particular volumes fill the pipette tip.
    d. If you notice air space at the end of the tip or air bubbles within the sample in the tip, carefully expel the sample back into its supply tube.

Expel the sample into a reaction tube or parafilm:
    a. Touch the tip of the pipette to the inside wall of the reaction tube/parafilm into which the sample will be emptied. This creates a capillary effect that helps draw fluid out of the tip.
    b. Slowly depress the plunger to the first stop to expel the sample. Depress to second stop to blow out the last bit of fluid. Hold the plunger in the depressed position.
    c. Slide the pipette out of the reagent tube with the measurement plunger depressed, to avoid sucking any liquid back into the tip.
    d. Manually remove or eject the tip into a beaker kept on the lab bench for this purpose.

Repeat with 50 µl and then with 250 µl.

Exercise III

Selecting proper micropipettors for measurements:
a) Identify proper micropipet and sketch what the dial should look like to measure each of the following volumes: 1.5 µl, 300 µl, 17.3 µl.

Exercise IV

Additive measurements:
    a) To a sample tube add the following volumes separately, changing tips each time. 3 µl, 5 µl, 7 µl
    b) You should have added a total of 15 µl to the tube. To test your accuracy, set you pipettor to 15 µl and carefully withdraw the solution from the tube.
    c) Consider: Does a small volume of fluid remain in the tube? This indicates an overmeasurement. After withdrawing all fluid, is an air space left in the end of the tip? This indicates an undermeasurement. (The actual volume of fluid can be determined by simply rotating the volume adjustment to expel air and push fluid to the very end of the tip. Then, read the volume directly.)
    d). If several measurements were inaccurate, repeat the exercise to obtain nearly perfect results.

Exercise V

Volume comparisons:
a) Practice measuring 1.0 µl, 5.0 µl, 10.0 µl, 50.0 µl, 100.0 µl, 500.0 µl. On an index card release the drops and visually compare the sizes. Draw the actual size of each droplet on a piece of paper.
b) Put one drop from an eyedropper or plastic transfer pipet on the index card away from your other drops. Estimate its size in µl __________.

Exercise VI

Loading Gels:
a) Practice loading diluted food coloring into wells of synthetic gels. Load 20 μL of diluted food coloring into each well.