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4 Steps to Master Reverse Pipetting

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What is reverse pipetting? Do you know how it can help you pipetting certain types of samples?

First for those who are not familiar with it. Pipettes have two stops. The first is generally used to aspirate liquid into the pipette, and the second stop ensures the entire volume is purged.

About Reverse Pipetting

Reverse pipetting is a useful technique for pipetting viscous liquids such as glycerol, mercury, detergent, honey or volatile solvents such as chloroform or hexane. Reverse pipetting mode will improve precision when working with extremely small volumes or liquids with a tendency to foam when pipetted.

Reverse pipetting can only be performed using an air displacement pipette. Basically, the pipette aspirates excess liquid and then dispenses only the desired amount, leaving a small amount of liquid behind in the pipette. During aspiration, an amount of liquid equal to the amount of purged air is added. This amount compensates for the liquid that remains as film inside the pipette tip during dispensing.

Reverse pipetting mode works well for the repeated filling of tubes or microplate wells with the same volume. The improvement in reproducibility will be noticeable. For repetitive pipetting using the reverse pipetting mode the excess liquid is kept in the tip as a buffer and not discarded until all the dispensing steps are completed. At that point, the residual liquid is discarded by depressing the plunger button to the second stop.

Using this method the tip is “automatically” pre-wetted but the extra liquid also helps when pipetting volatile solvents because some of the solvent will tend to evaporate into the air cushion. Be sure to hold the pipette vertically.

How to Perform Reverse Pipetting

When performing reverse pipetting depress the plunger button to the second stop and immerse the tip vertically into the liquid. Then smoothly release it, aspirating the liquid into the tip. In the next step, depress the plunger button to the first stop only dispensing the set volume of liquid into the receiving vessel. Withdraw the tip from the receiving vessel and then discard the residual liquid into a waste container by depressing the plunger button to the second stop.

Step #1) Push the piston down to the purge position (the second stop), then dip the tip into the liquid to a depth of 1 cm and draw the liquid up by slowing releasing the piston. Note that there is too much liquid in the tip at this point but when the liquid is dispensed by pushing the piston to the aspirate position (the first stop), the extra liquid is left inside the tip.

Step #2) Allow the plunger to move up smoothly to the rest position. Wait for all of the liquid to move up into the tip.

Step #3) To dispense the liquid into the receiving vessel by depressing the plunger to the first stop.  Be sure to touch the tip to the side wall of the container to take advantage of the elimination of surface tension through contact with the container which will ensure that the measured liquid being dispensed doesn’t remain in or on the tip.

Step #4) There will be liquid remaining in the tip. If the pipette tip is not to be re-used, depress the plunger to purge position over an appropriate waste container and then eject the tip. If the liquid is uncontaminated and going to be re-used, it can be pipetted back into the original container.

Reverse pipetting excels with viscous samples or repetitive dispensing of identical volumes. When pipetting fluids of low viscosity, be sure to calibrate your pipette for reverse pipetting to avoid dispensing more than the desired volume. However, if you can’t use reverse pipetting when manipulating viscous liquids be prepared for under delivery due to liquid retention in the pipette’s tip. Reverse pipetting is another pipetting option to consider going forward with your samples. Give it a try!

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About the Author : Jackie Williams

Jackie has a dual role at WHEATON. She serves as both a Product Manager for Liquid Handling and a Content Strategist for all WHEATON products. She has worked in the pharmaceutical, food & beverage and chemical industries prior to joining WHEATON. Her educational background includes a B.S. in chemistry from Ursinus College and an MBA from Rutgers University. Jackie brings products to market with insight gained from hands-on experience.

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