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Micropipetting basics?


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#1 azrael201

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Posted 01 February 2011 - 08:38 AM

Hi guys,

This is really dumb but i never realized how important this is until lately when my protein assay standards were looking funky. i was wondering if there were any tips people could give me on how to be good at micropipetting. i thought it was a no brainer but looks like it really is an art. i've been taught before to just submerge the pipette tip in the larger volume of solution to dispense smaller volumes like 1ul; however, it seems it is easy to double that volume from the extra sample that may be on the outside of the tip.

My current understanding is to place the tip of the pipette against the wall of the microfuge tube and expel the contents slowly and draw back. this has worked for the most part except when the liquid instead of forming a perfect bead at the tip flows backwards under my pipette tip and forms a pool. when i try to drag it off it does follow my tip for a while before it dislodges. I can't imagine all the extra volume i may have lost just observing how the sample sticks to my tip.

How can i prevent this? i want to believe as long as i am consistent with how i treat each sample it should be fine but seriously i can't help dragging circles with the tip and re-expeling the pipette to force the sample off.


Thanks!

Edited by azrael201, 01 February 2011 - 08:38 AM.


#2 hobglobin

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Posted 01 February 2011 - 09:15 AM

Pre-rinsing of the tip might help. Actually small volumes I also pipet directly in the larger volume, and for my experiments it works fine (though I don't have to pipet liquids for calibration curves).
I think it also depends on the liquid (surface tension, protein content, polarity) and wettability of the tips, there might be some differences between companies.

Gilson has a quite good instruction manual for their pipets (see page 5 ff). The link opens a pdf from Gilson (800kB).

Edited by hobglobin, 01 February 2011 - 09:17 AM.

One must presume that long and short arguments contribute to the same end. - Epicurus
...except casandra's that belong to the funniest, most interesting and imaginative (or over-imaginative?) ones, I suppose.

That is....if she posts at all.


#3 azrael201

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Posted 01 February 2011 - 09:30 AM

Pre-rinsing of the tip might help. Actually small volumes I also pipet directly in the larger volume, and for my experiments it works fine (though I don't have to pipet liquids for calibration curves).
I think it also depends on the liquid (surface tension, protein content, polarity) and wettability of the tips, there might be some differences between companies.

Gilson has a quite good instruction manual for their pipets (see page 5 ff). The link opens a pdf from Gilson (800kB).


Wow thanks that is a great manual. simple and to the point. I guess I am just confused about the pre-rinsing part? What does that mean? It sounds redundant unless I don't understand it is.

#4 azrael201

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Posted 01 February 2011 - 09:35 AM

never mind i read up a few pages and they explained "prerinse". thanks again!

#5 hobglobin

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Posted 01 February 2011 - 09:45 AM

It's for the first use of the tip, if you use it several times e.g. for a dilution series, as the first tip is "clean" and surface has to be wetted first and some parts of the fluid remain on the tip surface...i.e. the first pipetting with that tip has a higher loss than the next.
As the surface then is different (e.g. also polarity) you might get rid of the droplet easier...but that's just a guess.

One must presume that long and short arguments contribute to the same end. - Epicurus
...except casandra's that belong to the funniest, most interesting and imaginative (or over-imaginative?) ones, I suppose.

That is....if she posts at all.


#6 rhombus

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Posted 02 February 2011 - 12:59 AM

The only way to improve your technique is the old tried and tested way......

Sit down for an hour and practice your pipetting ONTO A PRECISION BALANCE.

You will instantly see with your own eyes IF you have pipetted the correct amount.

It is very important to do this as you can improve:

PRECISION AND ACCURACY.

This is an imperative skill to master as experimentation is dependent upon both of these skills.

Hope this is useful......there are no short cuts to excellence.

Kindest regards

Uncle Rhombus (34 years in the Lab)

#7 azrael201

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Posted 02 February 2011 - 12:29 PM

what is a precision balance?

i would not mind honing such a important skill especially with the reagents that are stickier. especially something like tween20 how do you even know you have the amount you want or should you just avoid pipetting tween20?

#8 hobglobin

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Posted 03 February 2011 - 09:58 AM

Rhombus is of course right, in the heat of the moment I forgot to mention this useful and tested method, that trains you the best way...
And a precision balance (scale) in an instrument for weighing tiny masses, usually the weighing range is around 0.1 mg.

One must presume that long and short arguments contribute to the same end. - Epicurus
...except casandra's that belong to the funniest, most interesting and imaginative (or over-imaginative?) ones, I suppose.

That is....if she posts at all.


#9 azrael201

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Posted 03 February 2011 - 10:11 AM

unfortunately the precision balance in my lab is broken...sigh

any other ways? i think i read one with pipetting 1 ul ten times and drawing it up with a 10ul pipette...

#10 knuf

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Posted 04 February 2011 - 06:30 AM

By working with rotation students I have found that doing a standard curve Bradford assay provides a good way of telling how good their pipetting skills are and provides some practice.

#11 Kaioshin

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Posted 13 February 2011 - 07:45 PM

what is a precision balance?

i would not mind honing such a important skill especially with the reagents that are stickier. especially something like tween20 how do you even know you have the amount you want or should you just avoid pipetting tween20?



Don't pipette Tween or Glycerol, weigh it




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