Three weeks into the new term and we have been going through the basic skills required to make accurate dilutions using automatic pipettes. Using a range of microbiological dyes, UTC students from Y10-13 are coming to terms with the "reliability" of these pipettes for delivering microlitre volumes in order to produce high quality standard curves using the Beer Lambert Law. The main aim of these experiments is to develop ownership of your experimental results and to keep practising until you get a result that is "trustworthy" and one that is unequivocal. At this stage on your journey, the most important thing is to ensure you understand the significance of each step in a protocol and that you appreciate when to use (in this case) a P1000, a P20 etc in preparing dilutions or reaction mixtures for PCR etc.
My one comment is that pipetting 1 microlitre quantities and diluting with 999 microlitres is likely to be a less accurate way of making a 1000-fold dilution than making two or more dilution steps. For example, mix 100 microlitres of your concentrate with 900 microlitres followed by a second dilution of say 10 microlitres of the first dilution with 990 microlitres of water. Also, the P5/P10 pipettes are mainly used when you have no choice i.e. when you are pipetting valuable, sterile or possibly hazardous samples. Finally, when you see data points that fall off an expected line, as top left (in the current classes, the absorbance versus concentration plot), simply repeat the measurement together with one or two of the samples that fall on your expected line. It is good practice to plot all measurements and not just the mean value. Key Words: Pipette, Gilson, standard curve, dilution, volumetric.
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