Pipetting

Operating micropipettes and pipette helpers are essential skills for working in molecular biology research. Both are required for laboratory exercises covered in this book, so you must be able to use them effortlessly. This section provides instructions on operating them and may refresh your memory if you have previously learned about them.

I. Micropipette

This video clip entitled “Using Micropipette by the University of Leicester” demonstrates how to use a Gilson Micropipette.

Micropipettes are used for accurately measuring tiny volumes of liquid, typically < 1 ml. Three micropipettes are required for the laboratory exercises described in this laboratory book (see below), plus one for volumes of 0.5–10 µl used occasionally. Each micropipette precisely measures a defined volume range. Although the volume range may vary slightly among different brands of micropipettes, the overall features are similar. The example here is from Labnet International Inc. For other brands, please refer to the manufacturer’s instructions for specific details.

Setting micropipettes to the desired volume may be confusing at first. It is common for beginners to be confused by the SL 20 and SL 200, as they both use the same size pipette tips. Remember to check before pipetting. For example, you may accidentally measure 50 µl instead of 5 µl or vice versa because of using the wrong micropipettes. You should check before drawing in the solution. When using micropipettes, always keep significant figures in mind. Since there are variations between brands of pipettes, most protocols refer to a volume without indicating the exact number of significant figures. You should always set your pipette to the correct volume with as many significant figures as possible. For example, suppose a protocol instructs you to pipette 20 µl. In that case, you should use your SL 20 pipette and set it to 20.0 µl (+/– 0.1 µl) instead of using the SL 200 and setting it to 20 µl (+/– 1 µl). Carry the correct number of significant figures through any required calculations. Since each micropipette has three numbers to set up the volume, different number combinations represent different volumes with different micropipettes (see table below). The following examples and instructions will help you understand how to set up the micropipettes until you feel comfortable setting them up yourself in the lab. Also, check with your laboratory partner if you are uncertain.

The volume range dispensed by each pipette is indicated on the side/top of the instrument and is in µl increments (0.5–10, 2–20, 20–200, 100–1000 µl). Do not dial past the lower limit (0.5, 2, 20, or 100 µl) or the upper limit (10, 20, 200, or 1000 µl) shown on the pipette. Be familiar and comfortable with these settings. Hold the pipette in one hand. Use the other hand to turn the volume adjustment knob until the required volume shows on the digital indicator. If you pass the desired setting, turn the dial a couple of revolutions above the desired setting and then dial down to the desired volume for maximum accuracy. Use the lock ring to lock the volume selector in place. When you’re ready to fill and dispense the micropipette, follow the instructions below:

1. Always hold the pipette vertically.
2. Always attach a new disposable tip to the pipette shaft every time you dispense a sample. Press firmly, not tapping, to ensure a positive airtight seal.
3. Depress the plunger to the first stop. This part of the stroke will take up the calibrated volume displayed on the digital volume indicator.
4. Immerse the disposable tip in the sample solution.
5. Allow the push button to return slowly to the up position. Never let it snap up.
   1. Take a moment to ensure that the total volume of the sample is drawn into the tip.
   2. Wait another second before removing the tip from the sample to allow the introduced liquid to enter the pipette tip fully. Removing the tip too quickly from the solution may result in air occupying some of the calibrated volumes.
6. Withdraw the tip from the solution. If any liquid remains outside the tip, gently touch it to the inside edge of the vessel to remove the excess sample liquid.
7. Ensure there are no air bubbles and that the volume corresponds to the desired volume. Look at the tip and the liquid in it!
8. To dispense the sample, touch the tip end against the inside wall of the receiving vessel and depress the plunger slowly to the first stop. Wait about two seconds (longer for viscous solutions), then press the plunger to the second stop (bottom of stroke), expelling any residual liquid in the tip.
9. Keeping the plunger fully depressed, carefully withdraw the pipette from the vessel, with the tip sliding along the vessel’s wall. Allow the plunger to return to the up position.
10. Discard the tip by pressing the tip ejector button. To prevent sample carryover, a fresh tip should be used for each sample.

To give you a sense of the expected volumes, pipette 500 µl, 100 µl, 50 µl, 15 µl, and 1.5 µl of water onto a small piece of Parafilm after selecting an appropriate pipette for each of the volumes. Ensure your instructor reviews your results as soon as you complete them.

II. Pipette helper for serological pipette

Although using electronic pipetting controllers/aids are another option, here we focus on the mechanical pipette helper for serological pipettes, which controls the filling and dispensing of liquids from pipettes with volumes ranging from 1 ml to 25 ml. It consists of a suctioning bulb to draw liquid into the pipette, a lever to control filling and dispensing, and a discharge (blow-out) button for expelling the remaining liquid in “blow-out” style pipettes. Inside the helper is a hydrophobic filter that prevents liquid from accidentally entering the helper. However, drawing liquid into the filter clogs it, requiring the replacement of the filter. The two most common mistakes that draw liquid into the filter are: a) drawing liquid up too quickly, especially in small volumes (e.g., 1 ml pipettes), or b) allowing the liquid in the pipette to drain back into the filter by not keeping the tip of the pipette down and below the liquid level.

1. Obtain a pipette helper, a 5 ml pipette, and a beaker filled with water.
2. Place the 5 ml pipette in the holder in the nose cone. You do not need to push hard. Rotate the pipette so that the volume markings are visible.
3. Squeeze the helper bulb to expel air. Do not squash it flat, as that will quickly wear out the bulb attachment to the helper’s body.
4. Place the pipette tip in the water and press up gently on the control lever. Stop when you have more than 3 ml in the pipette.
5. Press down gently on the dispensing lever until it reaches the 3 ml mark.
6. Place the tip of the pipette in the test tube with the tip touching the side.
7. Dispense the 3 ml into the tube and press the little bulb on the pipette holder to get rid of the last drop of liquid in the pipette. When no more liquid drains from the pipette, pull away gently from the side.