Pipetting methods and techniques for ELISA

Two types of pipetting methods are generally used for ELISA assays, standard and reverse pipetting. Please note that not all pipettes are capable of reverse pipetting, refer to the instruction manual provided with your pipette for more information.

Standard and reverse pipetting

Standard pipetting is used for the preparation of sample dilutions. Reverse pipetting is used for the addition of diluted samples, controls and reagents.

Good pipetting technique is essential for accurate and consistent results across multiple plates. Always be sure to use the correct pipette and tip for the volume being transferred.

Pipetting technique

  1. Push down to the first resistance on your pipette and release slowly to draw up the calibrated volume of sample into the tip.
  2. Touch the side of the inside of the tube with your pipette to remove excess liquid.
  3. Ensure that you have the proper volume of sample in your tip.
  4. Dispense the liquid into the measured diluent by pressing down on the plunger to the second resistance. Try and avoid placing the tip too deeply into the sample diluent
  5. Eject the tip into a waste container when the liquid has been expelled

Note – If using a multichannel pipette and the wells are empty, position the tip into the lower corner of each well, making contact with the plastic. If wells contain liquid, position the tips above the liquid, making contact with the plastic.

Standard pipetting and sample preparation

  1. Put a new tip on a single channel pipette. Ensure that the pipette and tip are correct for the volume to be taken up.
  2. Press the plunger on the top of the pipette to the first resistance.
  3. Draw the calibrated volume into the tip by slowly releasing the plunger up. Try not to place the tip too deeply into the sample.
  4. Touch the tip off the side of sample container to remove any excess liquid on the outside of the tip.
  5. Dispense the sample into the measured diluent by pressing the plunger past the first resistance. Try not to place the tip too deeply into the sample diluent
  6. Eject the tip into a waste container once all the liquid has been expelled

Note – Samples in wells can be mixed by using a multichannel pipette, by pushing the plunger up and down 3-7 times.

Reverse pipetting using a multichannel pipette

  1. Put new, clean, sterile tips on the multichannel pipette. Ensure they are straight and tightly secured.
  2. Press the plunger past the first resistance and halfway to the second resistance
  3. Draw the liquid up into the tip in a slow motion by slowly releasing the plunger up. Try and avoid drawing any air bubbles in the tips.
  4. Check for consistency in the volume of pipette tips.
  5. Touch the tips on the edge of the reagent reservoir to remove excess liquid on the outside of the tips.
  6. Slowly dispense the liquid into the wells by pressing the plunger to the first resistance. Be careful not to splash liquid out of the wells and ensure there are no drops left on the tips.
  7. Eject the tips into a waste container once all the liquid has been expelled

Note- Reverse pipetting uses more reagent/volume