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Do you know about reverse pipetting? A survey

pipetting techniques accuracy

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17 replies to this topic

Poll: Reverse pipetting survey (27 member(s) have cast votes)

You and reverse pipetting

  1. Never heard about it (until I read this) (8 votes [29.63%] - View)

    Percentage of vote: 29.63%

  2. I know what it is, but I never used it (3 votes [11.11%] - View)

    Percentage of vote: 11.11%

  3. I use it seldom or for special applications only (foamy, viscous samples) (13 votes [48.15%] - View)

    Percentage of vote: 48.15%

  4. I use it even in non-special circumstances/all the time (3 votes [11.11%] - View)

    Percentage of vote: 11.11%

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#1 Trof

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Posted 28 July 2012 - 06:15 AM

Reverse air-displacement pipetting mode. Not so unknown technique, as it is mentioned for example in Gilson guide to pipetting and I believe any other guides, but how many people really know about it and know it can really help them?

First for those who are not familiar with it.

Pipettes have two stops. The first is used to to aspire in usual mode, and the second one is used to purge the whole volume out. This ensures the right volume.

But you can use it reversly to also achieve correct volume, but with some benefits (and disadvantages).
First you push the button to the second stop, aspire to the rest position, then you only push to the first stop to get the desired volume out, leaving the residue in the pipette.

As described in a pictures [1] - [3].

Posted Image
You can then re-aspire and repeat as you wish [4], until you decide to finish by complete purge to a second stop [5].

Pros
- avoids foaming
- more accurate than normal mode with viscous liquids since it compensates for the film forming inside tip (may be true for the foaming samples also)
- quicker in multiple distributions by eliminating extra pluger step

Cons
From the picture is obvious that you aspire more than you need and this amount is in my experience considerable. So..
- not suitable for samples/buffers of limited amount
- you need to be carefull with filtered tips, as the actual amount inside tip can become much bigger than the tip is designed to (unfiltered tips have a lesser risk of this, but probably it's possible to exceed it's designed volume too)
- the residue left in the tip after drawing off is more likely unusable and gets wasted (I personaly return it back, when I'm not touching samples)

So, really helpful mainly for repetetive washings of foaming stuff like in CHIP, pipetting of enzymes (including PCR) and others.

Now end of the necessary educational part, and please tell me your experience in a poll Posted Image
(and aditional unoficial poll... is this topic even worthy the time? Posted Image

Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon


#2 pito

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Posted 29 July 2012 - 02:55 AM

The avoid foaming part.. ok, I get that, but you can also avoid (or at least minimise) foaming if you use your pipette carefully.

What I wonder: would it really be possible to use this the entire time? I can imagine its hard to stop at the first stop , the easy part in using the second stop is that you can simple push it down untill it stops..
With this method, you have to focus more and "wait" for the first stop.. you need to be really carefully...==> which makes me wonder: is it really quicker.. I doubt it to be honest.

If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#3 hobglobin

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Posted 29 July 2012 - 03:06 AM

I'd avoid using this technique, because the usual way of pipetting is so internalised and done automatically that then I'd really have to focus what I do to avoid that the normal pipetting mode comes back. Then I'd be confused and a mixed and therefore incorrect mode would happen soon.
So only if I've to pipet glycerol what happens rarely.
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.

#4 Adrian K

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Posted 29 July 2012 - 04:26 AM

Usually, I use this technique to dispense my PCR master mix into multiple tubes before I add in my template.
Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.

..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...

"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong

"It's all just DNA. Do it."---phage434

#5 Curtis

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Posted 29 July 2012 - 05:06 AM

I use it when I'm adding 200ul of Bradford dye to my samples to avoid formation of bubbles.

#6 Astilius

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Posted 30 July 2012 - 12:16 AM

I aspirate viscous liquids very, very infrequently. I'm more likely to use reverse mode pipetting for volatile liquids.
To the last, I grapple with thee; from Hell's heart, I stab at thee; for hate's sake, I spit my last breath at thee.

#7 mdfenko

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Posted 30 July 2012 - 06:50 AM

a friend used to use it for viscous liquids. i'm more patient.
talent does what it can
genius does what it must
i do what i get paid to do

#8 Trof

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Posted 01 August 2012 - 08:56 AM

pito: Yes, you can avoid it by being very carefull, but that usually costs a pretty much time, also depends on how much foamy the solution is. If you have to wash 24 wells like 10 x times each (with the same tip, so no touching of the surface), with a VERY foamy stuff, it is markedly quicker and less frustrating to do it in reverse mode. It is, I tried both.

As for the second question.. I don't really expect the people to use it all the time, the option is there is simply to balance the normal distribution Posted Image

hobgoblin: It's not that difficult to remember this other way of pipetting once you get it in hand, it's like driving with a different car, if you get used to one and learn to use other, and if you have some experience with both, when you switch cars you switch the way of driving too.
Of course for one-at-a-time thing it would be more difficult to keep this in mind, but even I who is well known for an initial lack of craft, was able to get used to it quickly enough in my washing series Posted Image

Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon


#9 hobglobin

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Posted 01 August 2012 - 10:01 AM

hobgoblin: It's not that difficult to remember this other way of pipetting once you get it in hand, it's like driving with a different car, if you get used to one and learn to use other, and if you have some experience with both, when you switch cars you switch the way of driving too.
Of course for one-at-a-time thing it would be more difficult to keep this in mind, but even I who is well known for an initial lack of craft, was able to get used to it quickly enough in my washing series Posted Image


well if in the other car the first gear is down right instead of top left, you're right Posted Image
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.

#10 Trof

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Posted 01 August 2012 - 02:52 PM

Who knows, when you have cars with steering-wheel on the opposite side. I don't, I don't drive I fly Posted Image

Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon


#11 hobglobin

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Posted 02 August 2012 - 07:35 AM

flying with a helicopter to work? wow....that's a luxury life Posted Image
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.

#12 Adrian K

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Posted 02 August 2012 - 07:57 AM

@Trof: Good life... Posted Image I never know doing research can earn so much like Donald Trump...
Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.

..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...

"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong

"It's all just DNA. Do it."---phage434

#13 Astilius

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Posted 02 August 2012 - 08:35 AM

a friend used to use it for viscous liquids. i'm more patient.


Meaow! LOL
To the last, I grapple with thee; from Hell's heart, I stab at thee; for hate's sake, I spit my last breath at thee.

#14 Trof

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Posted 03 August 2012 - 04:46 AM

@hobglobin: @Adrian: I actually walk to work, because we can't afford a car. So I'm using cheaper, motorless planes in my free time Posted Image

Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon


#15 pito

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Posted 03 August 2012 - 06:34 AM

@hobglobin: @Adrian: I actually walk to work, because we can't afford a car. So I'm using cheaper, motorless planes in my free time Posted Image

Lucky you that you are able to walk to work.. always nice to live near your work.

If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.






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