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Posts Tagged ‘sterile’

Liquid handling with pipettes

In Lab equipment, Pipet, Water on February 16, 2009 at 12:14 pm

Hi,

Today I would like to speak with you about liquid handling in the lab. Majority of our reactions are performed in liquids. From culturing of the cells to the specific enzymatic reactions performed, all are done in liquids. This is why we need an accurate and easy liquid handling device. We ususally perform liquid handling with pipettes.

So what is a pipette? I am sure almost everyone saw a pipette. A pipette is a device that aspirates liquids in order to transfer it from one vessel to the other. You can have a good description about general topics here.

You can have a very-very good introduction in the history of the modern molecular biology pipettes from a video by Lim Leng Hiong.

So, let’s see what “Freshbrainz” tell us about pipets:

But what kind of pipettes do we use?

The most basic pipette is a single use plastic pipette. It is not very accurate, but you can transfer liquids from one tube to a different one.

single-use-plastic-pipet1

You can use it like this:

We have a simillar pipette, a glas pipette that we use less for liquid transfer, but for removal of liquids from tubes. Usually after centrifugation steps we have a pellet and a liquid supernatant. If we want to discard the supernatant in a carefull and accurate way we use these “Pasteur” pipets. More details about Louis Pasteur here and please see a video about his work here.

So here is one of our Pasteur type, glas pipettes:

pasteur-pipet

And here is how we use a Pasteur pipette:

Of course the majority of our work is done with the so called Gilson pipettes. As our friend Lim Leng Hiong explained you these were specially designed for molecular biology work.

Below is video you can see how we handle liquids with a Gilson pipette. Please pay attention to the two stops made with my thumb. The first stop is reached when we aspirate the desired volume, while the second stop when we dispense the liquid. There is a button which is used to remove the single use tip of the pipette. So, please watch carefully the demonstration:

We have traditionally three type of pipette tips and these are differntiated by their color.

The smallest volumes can be measured with the 2 ul (2 microliter) pipette. This pipette is considered accurate between 0.5 and 2 ul-s.  The same tip is used for the 10ul pipette. We use this for volumes between 2ul-s and 10 ul-s. These pipettes are marked with gray, as shown below.

gilson-gray

The tip used with this pipetes is here:

gray-tip1

The next type of tip has yellow color traditionaly so the pipetes are marked with yellow:

yellow-heads

The same rule: P20 should be used between 10-20uls P100 between 20-100uls and P200 between 100 and 200uls.

The same tip can be used for these three Gilson pipetes, namelly these ones:

yellow-tips

The third type of this pipete is traditionally marked with blue. This is the one ml pipete. We call it P1000 and use it between 200 and 1000uls. Below is the head and the tip used for it.

blue-head

blue-tip

With this set of pipettes you can perfom majority of molecular biology reactions in the lab in an accurate way. They are not cheap, the price of one pipete is in the range of hundered dollars. They are precision instruments, so usually each researcher has his own set to use. Please pay attention to this and never use someone else’s pipete set only she or he specifically alowed it to you.

You can have a look on the usage of these pipettes on the best tutorial I have ever seen, produced by the University of Leicester here:

OK but what other alternatives so we have?

We have two very usefull type of additional pipettes. One is called the multichanell pipete, you saw it on Lim Leng Hiong’s video, and the other is the repeater pipete.

The multichanel pipet we use can have 12 or 8 chanells and you can have a look on it here:

multichanell-pipets

The range of volumes you can dispense with it can be seen here:

multichanell-range

With these pipettes the volumes can dispensed can be adjusted in steps and not in a linear way. You can see the adjustment volumes for both type of multichanell pipettes here:

multichanel steps

And here you have two videos about their usage:

The second type of very important help in the lab is the so called repeater pipete.
This pipete is able to dispense the same volume from a reservoir in a serial way.

Here is how it looks like:
repeater-pipet

The good stuff about these pipettes is that it can be used with different type of tips and it automatically recognizes the type of the tip you are using.

Here are the tips we use in general:

repeater-tips21

You can see on the next figure, that depending on the tip used the pipete is showing eighter 20 or 100 uls in the same position 1.

repeater-head

Here is a short video about how to use it:

With these pipets you can work easily in the lab. The master, the queen of lab pipettes is for sure the pipeting robot. We use a Tecan Genesis for pipeting smal volumes (5uls) in a serial way (e.g on a 384 well plate).

Have a look on this pipeting device:

robotical-pipetor-tubing

In my next post I will come up with serological pipettes and the price of the water in the lab!

Stay tuned, and lat me know if you have any questions!

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Water in the Lab

In Lab equipment, Water on February 1, 2009 at 5:50 pm

Hi,

Before we make the first experiment we have to discuss about some trivialities that might be different in the lab than in the outside world.

For example: water. Everyone knows what water is and I don’t want to recapitulate again the basics. You can have a real good overview here.

We use water for plenty of applications in the lab. Some of them are not specific to the lab world. Here are some examples:

3kep

Of course we use water for various lab specific purposes. The most important of these purposes is to prepare various solutions. In order to control as much as possible how our solutions will work we need a realy pure water. Tap water although is considered as pure drink water contains plenty of soluble components like: ions, colloids particles and so on. This water can not be used to prepare solutions. We use it to wash dishes but even after dish washing all dishes has to be rinsed with ion exchanged water. Ion exchaged water replaced distilled water in the last decades and stands for water that contains almost no ions at all. Distilation was used earlier to evaporate and … water and by this procedure you can get rid of the soluble salts from the water. The procedure was simmilar to the destilation of alcohool in distileries like this. The ion exchange resins are able to bind the ions from the water and produce a water that has the same qualities as distilled water has.

But how do you know if a water is pure?

It was told that you shoud use your senses: like smell it, view it, taste it. A clean water should be clear, tasteless and should not smell. But this is not enough. The easiest way to measure the presence of ions in water is by measuring its electrical conductivity. Soluble ions in the water will allow electricity to pass through the water. A really pure water is having very low conductivity.

In our lab we have a special tap for central ion exchanged water:

ioncserelt

So don’t worget, after washing lab dishes, please rinse everything at least twice with the ion exchanged water from this tap!

Can we use this water for solutions?

In some cases we could. Nevertheless due to the fact that we process sensitive biological samples like DNA and proteins we do not use this water for solutions in a molecular biology lab!

In order to prepare water for solutions we use so called “MilliQ” water. We introduce the ion exchanged water into an apparatus which is filtrating it through a replaceble cartridge. This filtrated water is free from colloids, proteins, ions and is suitable to be used in regular molecular biology solutions. Of course not for all applications! We will discuss this later. Here is the instrument that is producing the “Milli Q” water:

millipore-filter

You will find the water for solutions right in in a plastic carboy (also called demijohn) like this:

mq

You can use this water for preparing buffers for gel electrophoresis, western blot and so on.

By sterilizing it, you can make sterile solutions for cell culture applications. Nevertheless I would stronglly recommend that you should filtrate these solutions through a 0.2 micrometer filter. Majority of infecting agents (from bacterial origin) are larger than 0.2 micrometers so a sterilizes and/or filtered solution should be OK for cell culture applications.

There are some special applications that need special waters.

Two of them are RNA applications and cell culture applications for immune studies.

1. RNA applications.

While DNA can be protected quite easilly by adding EDTA as a chelating agent to the solutions (by this you get rid of the soluble Mg and other ions and you block the activity of DN-ases) RNA can not be protected like this. RN-ases are everywhere and are destroing the free RNA. That means that we have to use a special water that has no active RN-ases. Earlier we used so called DEPC treated water. Now we we use so called “Nuclease free water”. Earlier we were buying it in small 25ml bottles like this:

prom-nfw

Now we buy it in larger quantities and alliquot it. We use this water as NFW (Nuclease Free Water):

ambion-nfw

As a rule: USE ALLWAYS YOUR OWN NFW!!! Mark it with your name, and put a date when you oppened the tube.

2. The second type applications when we need an even purer water are the immunologic studies. In these cases we need a water thet is free of LPS (bacterial lipopolysaccharides, or endotoxins). The water we use for these applications is called “Embryo water” although we do not use it for embryological manipulations, it is LPS free. It is very important to alloquote it only in endotoxin free tubes, like cell freezing sterile vials.

Here is our LPS free water:

endotoxinfree

So these are the water types in our lab. We will discuss about the price of our water types later!