Experiment 1 Use of Micropipettor and Spectrophotometer
Submitted by: 4 Bio – 1, Group 1
Abquina, Eldrin, Alansalon, Ana Loren, Ani, Donnallyn, Benedito, Ma. Ciara
Submitted to: Josefino R. Castillo, M.Sc.
Date Submitted: June 30, 2011
INTRODUCTION
Molecular biology is a specific type of biology that is concerned with the examination of cells at the molecular level and that focuses on the interactions between many cell systems. Over the past century, scientists have found an ever-increasing number of ways to probe the secrets of the cell and the biochemical processes that make life what it is. Biologists now have a wide variety of tools at their disposal to study the functions of genes and proteins and figure out how cells work. Laboratory instruments are devised to achieve accuracy and precision as it deals with up to microscopic level. Virtually any area of science that you enter will require that you master the skills of pipetting and many biological assays require the use of the spectrophotometer. There are many devices that laboratory workers use to pipette solutions. Pipettes are used to accurately measure and dispense small volumes of liquid. The capacity of a micropipette can range from less than 1µl to 1000µl (1ml), while macropipettes can measure volumes greater than 1ml. These are used to physically interact with microscopic samples, such as in the procedures of microinjection and patch clamping. Most micropipettes are made of borosilicate, aluminosilicate or quartz with many types and sizes of glass tubing being available. Each of these compositions has unique properties which will determine suitable applications. Micropipettes brands include: Eppendorf, Hamilton, Rainin, Drummond etc. Accuracy in pipetting is very important as the precise amounts of solutions in biological and chemical assays is critical, particularly when constructing standard curves by which the unknown or experimental samples are to be compared. A spectrophotometer is a device used to measure light intensity. A spectrophotometer can measure either absorbance or transmittance of light. A small beam of light with a specific wavelength is emitted from the spectrophotometer which goes
through the sample in a small glass container called a cuvette. The spectrophotometer measures how much light is absorbed by the sample or how much of the light passes through the sample which is transmittance. It has varied applications in the qualitative analysis of sample purity, DNA and protein quantitation, cell density measurements, and assays involving enzyme – catalyzed reactions. The most common spectrophotometers are used in the UV (200 – 400nm) and visible regions of the spectrum (400 – 700nm), and some of these instruments also operate into the near-infrared region (700 – 900nm) as well. The VIS and UV spetrophotometric techniques are used mainly on qualitative determination of components present at low concentration. The IR measuring technique is mainly used for qualitation. The spectrophotometer compares the intensity of the trensmitted light with that of the incident light. The objective of this experiment is to familiarize the students with the use of the micropipettor and the spectrophotometer and to some of their applications in scientific research. It also aims to determine the accuracy and the precision that the students and the instruments can generate.
MATERIALS AND METHODS
MATERIALS
Materials:
Special equipment:
GENESYSTM 10 uv-is
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Bromphenol blue (1.25%w/v)
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9 Microcentrifuge tubes
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Micropipettors and tips
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Semi-micro cuvettes
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Distilled water
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Vortex mixer
Micropipettor
Tips
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Spectrophotometer
Microcentrifuge Tubes
Vortex
Spectrophotome ter
Bromphenol Blue
Distilled Water
Cuvette
Figure 1 – Equipment and Materials
METHODS
A. Testing accuracy of operators
The spectrophotometer was warmed up for twenty minutes before using and was set at 540nm. An amount of 1 mL of distilled water was placed in each of five microcentrifuge tubes. With the micropipettors, successive amounts of bromphenol blue were added. The volumes of the bromphenol blue added were 0.5, 1.0, 1.5, 2.0 and 2.5 µL respectively. The instructions for use of these micropipettors were followed carefully. Each tube was then vortexed until the dye was in solution. The spectrophotometer was zeroed with distilled water. The contents of the microcentrifuge tubes were transferred to the cuvettes. After which the absorbance were read and the readings were recorded. Finally, the results were graphed. B. Testing precision of operators An amount of 1 mL of distilled water was placed in the test tube. With the micropipettors, 4.0µL of bromphenol blue was added by each member in each microcentrifuge tube. The tube was then vortexed until the dye was in solution. The spectrophotometer was zeroed with distilled water. The contents of the microcentrifuge tubes were transferred to the cuvettes. After which the absorbance was read and the reading was recorded. The standard deviation and the coefficient of variation were calculated, where: SD = N∑x2 – ∑x2N-N-1
CV = SDMean RESULTS AND DISCUSSION
There are two laboratory instruments commonly used in Cell and Molecular Biology namely the micropipettor and spectrophotometer. In this experiment, the following results were obtained:
Table 1 – Bromphenol Blue Concentration and Absorbance
Bromphenol Blue (μL)
Absorbance (540nm)
0.5
0.008nm
1.0
0.011nm
1.5
0.014nm
2.0
0.026nm
Standard Deviation
Mean = 0.082 SD = [N∑x2 – (∑x)2]N-(N-1)
= 0.03473 Coefficient of Variation
CV = SDMean = 0.42 Figure 2
Table 2 – Absorbance
Student
Absorbance (540nm)
Abquina
0.0024nm
Alansalon
0.0030nm
Ani
0.0027nm
Benedito
0.0020nm
Standard Deviation
Mean = 0.101 SD = [N∑x2 – (∑x)2]N-(N-1)
= 0.0148
Coefficient of Variation
CV = SDMean = 0.145
Figure 3 – Absorbance result per student trial
This experiment tackles on the familiarization of two commonly used laboratory equipments: the micropipettor and spectrophotometer. A specific amount of bromphenol blue was added and mixed with each of the microcentrifuge tubes containing 1mL of distilled water. Using the spectrophotometer, the absorbance for 0.5μL of bromphenol blue was 0.008 nm, for 1.0μL bromphenol blue it was 0.011nm, 0.014nm was recorded for 1.5μL of bromphenol blue, for 2.0μL bromphenol blue, 0.026nm and lastly for 2.5 μL bromphenol blue, 0.023nm.
The results show that there is a direct relationship between the amount of dye in the solution and the amount of light absorbed by the solution. As the amount of dye increases, the amount of light absorbed also increases. There was one significant error in the results. The fifth absorbance dropped rather than increase. This might be the result of mixing up the last two cuvettes or error in pipetting. Each of the students placed 2.5 micro liters of bromphenol blue in their respective micro centrifuge tubes and their solutions were mixed using the vortex mixer. The results varied from each student’s solutions that used the same amount of dye and micropipettor. Though the results varried the values obtained we’re really close to each other. This might be the effect of not changing the tip per trial of pipetting. For this experiment, we can conclude that the skills of the students with regards to the use of a micropipette and spectrophotometer can only improve. Being familiarized already with the equipment, the students can eliminate the possible casuses of error a graph of the amount of bromphenol blue versus the absorbance in the spectrophotometer should give a straight line. The standard deviation and coefficient of variation provides a measure of accuracy and reproducibility of the pipetting skills respectively; the smaller the standard deviation and coefficient of variation, the better the skills of the student.
REFERENCES
[1] Berkley, James. Use of Automatic Digital Micropipettor. USA. CNE. 1993 [2] Bissen, Shirley. How to Use a Micropipettor. USA. Prentice – Hall. 1998.
[3] Harris, Daniel. Exploring Chemical Analysis (4th ed). New York: W.H. Freeman & Company. 2009. [4] Pungor, Erno. A Practical Guide to Instrumental Analysis. USA: CRC Press LLC. 1995. [5] http://www.uvm.edu/~cbookwal/001B/Labs/exp2.pdf [Date retrieved: June 27, 2011] [6]http://faculty.weber.edu/nokazaki/Comparative%20Animal %20Physiology/Laboratory/Spectrophotometry04-1.pdf [Date retrieved: June 27, 2011]
