Biology Micropipetting

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Used a P100 to pipette 93.6 micro-litres of fluid. There was an air gap in the pipette tip the first time I did it but no air gap the second time.

I'm assuming there shouldn't be an air gap but can somebody tell me for sure?
 
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BillTre

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Do you mean a bubble in the tip after sucking up the fluid?

If it was a bubble and it all ejected, it probably measured the correct amount.

If you took the tip out of the fluid before it was done sucking up fluid resulting in a non-fluid area at the bottom of the tip, you would have the an incorrect amount.
 

epenguin

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Is this the first time you have ever used such a pipette? By 'air gap' do you mean between the liquid and the tip of the pipette?

The gap suggests that you withdrew the tip it out of the liquid before you had totally aspired. For all I know you may have 93.6 μL there, but the point is knowing whether you have. Certainly if I saw that I would be willing to invest an extra 10 seconds to do it with no gap. (It is many orders of magnitude more than 10 seconds since I was in a lab by the way).

The manufacturers give data on the accuracy of their products https://www.btlabsystems.com/P100_Pipette It looks like for your model they are claiming about a 1% inaccuracy. Surprises me a bit for a variable volume pipette, but as I say it has been a long time ... all three significant figures is too much therefore. Most of the time biochemists will go on blind faith on what their instruments tell them. Occasionally it is really important to check whether this faith is justified. In any case it is good practice to do so from time to time, I guess by weighing the drop. (I remember a paper published of someone who did this when Eppendorf pippettes were new, I think in Nature.) anyway if you do it without a gap every time then it will be the same every time and probably not matter.
 
23
2
Is this the first time you have ever used such a pipette? By 'air gap' do you mean between the liquid and the tip of the pipette?

The gap suggests that you withdrew the tip it out of the liquid before you had totally aspired. For all I know you may have 93.6 μL there, but the point is knowing whether you have. Certainly if I saw that I would be willing to invest an extra 10 seconds to do it with no gap. (It is many orders of magnitude more than 10 seconds since I was in a lab by the way).

The manufacturers give data on the accuracy of their products https://www.btlabsystems.com/P100_Pipette It looks like for your model they are claiming about a 1% inaccuracy. Surprises me a bit for a variable volume pipette, but as I say it has been a long time ... all three significant figures is too much therefore. Most of the time biochemists will go on blind faith on what their instruments tell them. Occasionally it is really important to check whether this faith is justified. In any case it is good practice to do so from time to time, I guess by weighing the drop. (I remember a paper published of someone who did this when Eppendorf pippettes were new, I think in Nature.) anyway if you do it without a gap every time then it will be the same every time and probably not matter.
Thanks for the reply. Yes, it is my first time using these pipettes, we just learned how to use them. The point of the experiment was to learn good technique and see if we were accurate. I did do it twice, the first I had an air gap and second time I did not. A few of my friends had air gaps as well. By air gap I mean after I was done sucking the liquid, in the tip, there was fluid, then like a gap (maybe air bubble), and then fluid again. I guess its safe to say I was inaccurate in the first trial?
 
23
2
Do you mean a bubble in the tip after sucking up the fluid?

If it was a bubble and it all ejected, it probably measured the correct amount.

If you took the tip out of the fluid before it was done sucking up fluid resulting in a non-fluid area at the bottom of the tip, you would have the an incorrect amount.
Thank you for the reply. By air gap I mean once I was done sucking the liquid, in the tip, there was fluid then like an “air bubble” then fluid again. The point of the experiment was to see if our micropipetting techniquie was accurate since its our first time. Is it safe for me to say I was inaccurate in my lab report?
 

BillTre

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Thank you for the reply. By air gap I mean once I was done sucking the liquid, in the tip, there was fluid then like an “air bubble” then fluid again. The point of the experiment was to see if our micropipetting techniquie was accurate since its our first time. Is it safe for me to say I was inaccurate in my lab report?
To me this sounds like a bubble that formed on the side of the pipette tip. This happens sometimes, perhaps due to that part of the tip plastic not being so wet-able as other parts so the water flows around it. This would have sucked up the right volume.

Alternatively you may have sucked up the fluid so quickly that a bubble was sucked into the tip along with the fluid and then stuck on the side. This would give an incorrect volume.

When you eject the fluid, there is a stop you can feel before you fully press the plunger. In ideal situations, this would fully eject all the fluid since it is the volume that was sucked in. Fully pressing the plunger pushes out more volume to ensure everything is ejected.
If you watch how much fluid comes out vs. when you feel the first stop, you can get an idea of whether you sucked up the desired amount or not, unless the air is ejected also.
 
Hi. Interesting post.... ... I'm coming from 35 years in a medical laboratory. Pipettes are my daily tool.


I will take this stepwise.


1. What is the liquid you measured/used.
2. The temperature.... both air and liquid.

Both these two can make the weight different to the volume. So let me put it this way....in theory deionised water at 23 deg (as well as particular air pressure, humidity and possibly other factors) will weigh lets say 1.000 grams per 1000ul. So your 100ul "should" weigh 0.100g. Understand.

3. The accuracy of the balance. Was the balance correctly calibrated.


In a laboratory to certify the accuracy of a pipette it is acceptable to have a tolerance of 10%. But it will also dependent on the use. We have some tests that require the pipettes to have a 2%tolerance. So if you dispense a liquid 100ul on the dial should give you a range of 90 - 110 (10%). Or ... in some of our pipettes 98 - 102.....


5. Now there are other variables that affect the volume...
Such as the correct tip and fitted correctly.
When aspirating a constant raising of the thumb part. As well as tge dispensing. Amazing how much difference this makes.
Pipette should be upright and maintained upright. .... try this next time aspirate a solution then tip your pipette about 45deg look at what it does.
Reverse pipetting or forward pipetting...... do you know the difference????. This is one of the biggest factors.

The O ring (new or used).
When was the pipette last calibrated and adjusted????

I think I've gone on too long...



To confirm what "volume" you did you need yo repeat tge exercise 10 consecutive time and record each one.


Sorry to babble on and on...

This is a big topic and I take each new graduate just on pipetting and liquid handling for over half a day. ..
 

atyy

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anyway if you do it without a gap every time then it will be the same every time and probably not matter.
Hmmm, surely it's better if you do it different everytime and get the same results - that way you will know the error doesn't matter for reproducibility of your results.

If you do it the same every time, then your results may not be reproducible by others, since you will be consistently doing something, but writing in your paper as something else. Of course, you can write that you should consistently pipette with an air gap ...
 

atyy

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Thank you for the reply. By air gap I mean once I was done sucking the liquid, in the tip, there was fluid then like an “air bubble” then fluid again. The point of the experiment was to see if our micropipetting techniquie was accurate since its our first time. Is it safe for me to say I was inaccurate in my lab report?
Yes, that sounds inaccurate to me. The fluid volume should be one continuous volume.


 
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17
3
Used a P100 to pipette 93.6 micro-litres of fluid. There was an air gap in the pipette tip the first time I did it but no air gap the second time.

I'm assuming there shouldn't be an air gap but can somebody tell me for sure?
So an old-style easy way to check your accuracy with any pipette is with water. The scientific system of weight is based on the weight of water. So 1 gramme = 1 ml of water.
All you need is some light tissue paper. Put it on a balance and zero it, and pipette, lets say 1 ml of water onto the tissue. Of course make sure the balance air bubble is within the circle so that you know the balance is level. If you get 1 gramme then your pipette is accurate. You can check all your pipettes and your technique this way ( 100uls = 0.1 grammes).
Basically I can get close to 100% accuracy.
 

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