Do you know about reverse pipetting? A survey - (Jul/28/2012 )

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>.


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
(and aditional unoficial poll... is this topic even worthy the time?

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.

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.

Usually, I use this technique to dispense my PCR master mix into multiple tubes before I add in my template.

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

I aspirate viscous liquids very, very infrequently. I'm more likely to use reverse mode pipetting for volatile liquids.

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

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

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

Trof on Wed Aug 1 16:56:39 2012 said:

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

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