BI0157 Biochemistry Labs

How to use the automatic pipettes

Goals of the experiment

The aims of this experiment can be summarised as follows:

• To become acquainted with the laboratories and safety routines

• Gain experience in experimental design

• Practise pipetting and the use of spectrophotometers

To get access to the course laboratories you will have to know your way about them, and know how to use a few instruments. There are also certain routines which facilitate the experiments and working in the lab. After succesful completion of this lab will you be allowed to use the VÖL laboratories.

This introductory practical consits of three parts. The first part is a general introduction to the facilities of the lab. It includes orientation and safety. During the second part you will learn how to use micropipettes and the spectrophotometers by developing your own assay for determining hemoglobin concentration. Finally, we will go through the guidelines in "General instructions on how to write a lab report" which has been handed out as part of the lab compendium.

Orientation

You will get to know where to find the ice-machine, refrigerator, freezer and -80 freezer, just as the cold-, microscope-, spectrophotometer-, and electrophoresis-rooms. Also where solutions and chemicals are stored and other storage rooms.

Further you will be shown where to find hoods, incubators, water baths, and also learn about different water qualities. You will after this know where to put dirty, broken, radioactive or otherwise contaminated glassware.

Safety

You must know:

• where to find emergency eye-wash and showers, and how they work.

• how the hoods work and how to vary the flow.

• where to find the fire extinguisher, fire blankets and First Aid kit.

• how to handle dangerous chemicals & solutions.

• what to do in case of emergency.

Eating, drinking, smoking etc. in the lab is absolutely forbidden. You should always be wearing a proper lab coat inside the lab. Never wear your lab coat outside the lab. (Why?)

When dealing with acids and bases you should be wearing latex protective gloves. When working with very cold or very hot or fuming substances, special protection measures particularly for your hands and eyes should be taken.

Spectrophotometry

Spectrophotometry is a good way of determining the concentration and sometimes the purity of certain solutions. The test solution is put in a plastic, glass, or quartz vessel called a cuvette, and placed before a source of monochromatic light. There is a detector at the other end of the cuvette which measures the amount of light transmitted through the cuvette.

Two sides of the cuvettes are transparent and these are the sides through which the light beam should pass. Never touch these sides with your fingers or any other material, (Why?)

The solution has to be up to a certain height in the cuvette to make sure the whole light-beam passes the sample.

The length of the path of the light through the sample should always be the same (1 cm). In case multiple measurements have to be made, always make sure that the same end of the cuvette faces the light beam. There is a small arrow marked on the cuvette to indicate the direction.

Remember to mix the contents in the cuvette if more than one solution is added. This is easiest done by either covering the cuvette with its stopper or some parafilm and gently turning it upside-down a few times, or by gently pipetting the contents in and out.

If you use the same cuvette to measure different concentrations of some reagent or mix your solutions with the same tip or rod you should always proceed from low to high concentration. (Why?)

Do not allow air bubbles to get trapped inside the cuvette during measurements. (Why?)

When working with UV-light, quartz cuvettes have to be used, since glass or plastic cuvettes absorb UV light. Also remember that the UV-lamp has to be on a few minutes before it gives a stable light.

The concentration of a sample can be calculated from Lambert-Beer's law:

A = e * c * l

Where A is the absorbance, e the extinction coefficient, c the concentration, and l the length of the beam in the cuvette (1cm).

For best accuracy in the measurements, adjust the concentration of your samples so that 0.01 < A < 2.00. (Why?)

Pipetting

The tip has to be fastened firmly to achieve the right volume. Always change the tip between every new solution, or if it has become contaminated.

Never release the button on the top of the pipette without control from your thumb. Otherwise, some solution from the tip may be sucked into the pipette contaminating it.

Avoid touching the tips with your fingers. To dispose a used tip use the appropiate button on the pipette. Use a beaker or a plastic can to gather disposed tips on your lab bench. At the end of the lab you can put all the tips in the waste basket.

If the solution you are pipetting is toxic/radioactive, put tips in the special box for toxic/radioactive waste.

Never try to take a bigger volume than the pipette is made for. Always keep the pipette in an upright position, when you are finished with a pipette put it back on its holder (don’t leave it lying down on the table!). This is to avoid pulling solution into the actual pipette.

Use the pipettes in their appropriate measuring region. This will improve the accuracy.

Practical Exercise

How to use the automatic pipettes

In biochemistry and molecular biology laboratories often very small volumes, between 0.1 µl and 1000 µl, are pipetted. The aim of this exercise is to become acquainted with the automatic pipettes that normally are used for this purpose. It is important that you know how to use these pipettes accurately and to practise this you will pipette a series of dilutions from a CuSO₄ -solution of known concentration. Subsequently the samples are measured in the spectrophotometer and finally you should draw a graph with A⁶⁰⁰ as a function of the CuSO₄ -concentration.

Materials:

Pipettes

20 µl (For volumes between 2-20 µl),

200 µl (For volumes between 20-200 µl)

1000 µl (For volumes between 100-1000 µl