I'm doing PCR and qPCR frequently. My assay is extremely sensitive so I need to pipette a very exact volume from my SOP. From my experience and also my school lab course, I learned to press the pipette to the 1st stop to get the desired volume, and then press all the way to the second stop to expel all the liquid. However, I was harshly judged by my supervisor for using such skill. My supervisor said it must be done only at 1st stop, picking the liquid at 1st stop and expelling the liquid at 1st stop. I tried the above way but always left some residue on the wall of the tip, which raised my concern a lot.
My supervisor made a judgment that if I can't expel liquid at the 1st stop, it is my tech skill problem. I also asked some co-workers ( sophisticated), and some of them indeed stop at the 1st stop to dispense an accurate amount, and ignore the residue on the tip. One coworker advised because of the air pressure, angle, and speed when you pick the liquid, it might over-pick the liquid so dispensing at 1st stop is accurate enough.
My liquid usually thawed cold serum, enzyme, probes, and buffer, so I'm less concerned about evaporation. I'm grateful for any suggestions here!
I had the same problem when first learning qPCR. Although I was also concerned about the solution left behind in the tip, in an 11uL total reaction volume, I found that the only way I was able to achieve consistent CT values across multiple plates was to only go to the first stop when dispensing solution. my CT values in general became slightly higher but were much more consistent. I began doing this based on the suggestion of a colleague who also dispenses solution down to the first stop for qPCR.
Hope this helps!
When pipetting, it's generally recommended to press the pipette to the first stop and then dispense the liquid at the same stop for accurate results. However, some liquids may leave residue on the wall of the pipette tip depending on the liquid properties or the technique used during pipetting. To reduce the amount of residue, I would suggest trying the following:
Ultimately, I recommend you follow the technique recommended by your supervisor or laboratory protocol for consistent and reproducible results.
Good luck!
Hello Yun Sawa
"I learned to press the pipette to the 1st stop to get the desired volume, and then press all the way to the second stop to expel all the liquid."
You are right. The method you are following is called forward pipetting.
There are two types of pipetting techniques.
Forward and reverse pipetting modes that can be used with a mechanical pipette.
In forward pipetting, the target volume is aspirated and dispensed, and a separate blowout step is used to completely empty the tip by pressing the plunger to the second stop.
In reverse pipetting, the target volume is aspirated with an excess amount. Upon pressing the plunger to the first stop, the total target volume is dispensed, and the excess amount is left in the tip. The excess amount is either returned or discarded by pressing the plunger to the second stop. The presence of the excess volume has significant benefits for pipetting performance in certain circumstances, such as when one has to pipette volatile or viscous liquids.
You should go for forward pipetting. In forward pipetting, you may depress the plunger to the first stop, immerse the tip into the liquid, and aspirate by releasing the plunger. Remove the pipette from the liquid and depress the plunger to the second stop to dispense the entire content. While dispensing the sample, position the tip to touch the side of the container to deliver any residual sample remaining in the tip. Keep your thumb pressed on the second stop of the plunger and remove the tip to avoid sample re-aspiration into the pipette tip. Make sure that you see the sample leaving the tip. The forward mode of pipetting yields better accuracy and precision.
Besides the above, some tips of pipetting are given below which may be of help to you.
1. You should always pre-wet the tip as it increases humidity within the tip, thus reducing any variation. If you are using the tip multiple times without pre-wetting it, then there are chances that a lower volume will be dispensed in the first few rounds.
2. Apply the same pressure and speed when you aspirate and dispense the contents for reproducibility.
3. When you are pipetting small volumes such as less than 50ul remove the pipette from the center of the vial. Avoid holding the pipette at an angle as it may alter the volume of the sample aspirated.
4. When you are about to dispense the content of the tip, you may observe some droplets on the outside of the tip. You may wipe out those droplets with a lint-free cloth. But be a little careful as excess wiping may suck the sample from the opening of the tip leading to sample loss.
Best.
Actually I think you were right about "press all the way to the second stop to expel all the liquid", and this was the way that is instructed by multiple pipette manufacturers: https://handling-solutions.eppendorf.com/fileadmin/Community/FAQs/2112017/PDFs/Poster_PipettingTechniques_72923.PDF
https://lab.plygenind.com/how-to-use-pipette-correctly
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