_this exercise and explanation was modified from the Putnam Lab pipette exercise, which they modifed from somewhere else

Proficient pipetting is very crucial in this lab, for three reasons:

  • Accurate pipetting is crucial to the success of molecular projects. Mistakes during pipetting may cause your experiments (e.g. PCR, DNA isolation) to fail or to be irreproducible, and thus cause long delays and considerable expense
  • Pipettes are delicate pieces of equipment with high accuracy, which can easily be knocked off their calibration. Furthermore, they are expensive – each set of pipettes represent about £ 1,500.
  • Pipettes are the main source of contamination e.g. in PCRs, and thus can cause a lot of problems.

Never

  • Never rotate volume adjustor beyond the upper limit
    • Changes calibration
  • Never use without a tip.
    • Liquid will get into piston – damage and contamination.
  • Never lay down or turn upside down a pipette with filled tip.
    • Liquid could run back into the piston, and damage/contaminate it.
    • Use pipette holders provided
  • Never let plunger snap back after withdrawing or ejecting fluid
    • Damages piston
  • Never use other person’s pipette without letting them know
    • To track contamination sources

Use

  • Use the correct pipette. Never adjust the volume beyond the maximum setting, but also do not use pipettes that are too large. Accuracy and precision drop rapidly towards the lower limit of settings.
  • Adjust volume by turning the volume adjustment knob or the plunger. Be sure to locate the decimal point correctly when reading the volume setting (see above). Always dial DOWN to the desired volume to avoid mechanical backlash affecting accuracy.
  • Firmly seat the proper-sized tip on end of the pipette. If the fit is lose, you will draw up less volume than intended and the liquid will drip from the tip during use.
  • When withdrawing or expelling fluid, always hold the tube firmly between thumb and forefinger. Hold close to eye level to observe change in fluid level in tip. Do not pipet with the tube in rack or when someone else is holding the tube.
  • The pipettes have a two-stop position plunger. Depressing to the first stop measures the desired volume. Depressing to the second stop introduces an additional volume of air to blow out any remaining fluid from the tip.
  • To withdraw fluid from tube.
    • Hold the pipette almost vertically (< 20o from vertical)
    • Depress plunger to first stop and hold. Dip tip 2-4 mm into the fluid. Do not push down to the bottom of the vial, otherwise the tip is blocked and you draw less liquid than intended.
    • Gently release thumb. If you release quickly, you will create aerosols (small droplets), which will contaminate the pipette.
    • Wait for a second or so, to confirm that all the liquid has been taken up (especially important for high viscosity liquids).
    • Slide pipette tip out along wall of tube to dislodge remaining droplets adhering to the outside of the tip.
    • Check that there is no air space at the very end of the tip.
    • Learn the approximate levels that particular volumes fill the tip – this will allow you to check your pipetting visually.
  • To expel sample into tube
    • Touch tip to wall of tube or to the liquid at the bottom of the tube if there is already some in there
    • Slowly depress plunger to first, and then to the second stop to expel fluid.
    • While keeping the plunger at the second stop, slide the tip out of the fluid, making sure you don’t suck up any liquid and that there is no liquid still left in the tip.
  • Eject the tip into trash, by pressing the tip ejector button.

Prevention of cross contamination

  • Use a fresh tip each time
  • Do not touch the tube with the pipette, only with the tip
  • If you suspect pipette-body contamination, wipe with ethanol and 10 % bleach solution on the outside.
  • Draw up liquid slowly to prevent the formation of aerosols, bubbles also mess up your volume
  • Use filter tips to reduce contamination risk if you think the step requires it, they are expensive so use them when really needed
  • Use the correct tip size

Pipetting exercise

Be careful to use the correct pipette. Use distilled water. You don’t have to change tips for these exercises. As you do these exercises, look at the pipette tip when it is holding a volume so you can learn to check your pipetting visually.

P1000 (200-1000 µl) – 7 eppendorf (1.5mL microcentrifuge tubes, they have a lot of names) tubes:

  1. Add 1000 µl to three eppendorf tubes.
  2. Visually check that each tube has the same volume.
  3. Remove 750 µl from each tube and pipette into three new tubes.
  4. Visually check that each tube has the same volume.
  5. Remove 250 µl from the original three and pipette into one new tube.
  6. Remove 750 µl from this tube and discard pipette tip.

P200 (20-200 µl) – 3 sets of PCR 8 well strip tubes, and 1 eppendorf tube:

  1. Add 100 µl to each well of a set of strip tubes.
  2. Visually check that each tube has the same volume.
  3. Remove 50 µl from each tube and pipette into a new set of strip tubes.
  4. Visually check that each tube has the same volume.
  5. Remove 30 µl from the original strip tubes and pipette into a new set of strip tubes.
  6. Visually check that each tube has the same volume.
  7. Remove 20 µl from the original strip tubes and pipette into one new tube.
  8. Remove 160 µl from this tube and discard pipette tip.

P20 (10-20 µl) – 1 set of strip tubes and 1 eppendorf tube:

  1. Add 18 µl to each well of a set of strip tubes.
  2. Visually check that each tube has the same volume.
  3. Remove 12 µl from each well and pipette into one new tube.
  4. Remove 96 µl from this tube and discard.

P20 (10-20 µl) – 1 set of strip tubes:

  1. Add 7 µl to three 0.2 mL tubes.
  2. Visually check that each tube has the same volume.
  3. Add 2 µl to each tube.
  4. Visually check that each tube has the same volume.
  5. Remove 9 µl from each tube and pipette into three new 0.2 mL tubes.
  6. Visually check that each tube has the same volume.

Large volume:

  1. Add 179 µl to a tube
  2. Add 465 µl to the same tube
  3. Remove 602 µl from the tube
  4. Remove 42 µl from the tube

Small volume:

  1. Add 8.3 µl to a tube
  2. Add 13.5 µl to the same tube
  3. Remove 15.6 µl from the tube
  4. Remove 6.2 µl from the tube

After mastering these exercises with water, try the large and small volumes with glycerol. EXTREMELY viscous

How accurate were you?

  • Is some liquid left in the tube? Measure it by drawing it up, and adjusting the pipette volume until all air below the liquid is expelled from the tip. Remember to hold the pipette vertically while doing so.
  • Did you draw air up with the last removal of liquid? Measure the volume of step 4 by adjusting the volume downward. Remember to hold the pipette vertically while doing so.

The pipettes have an error on accuracy and precision of about 1% - any more than that is human pipetting error! If you got it very wrong, try again.

Always calculate an extra volume for each reagent due to the pipetting error