QBM LAB Report 1 - ertert PDF

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Lab Report 1: Micropipetting, Centrifugation, and Spectrophotometry Author: Amanda Hollingsworth QBM Laboratory, Section 87 January 20, 2021

Objective: The objective of this laboratory is to familiarize oneself with the equipment and the techniques used in the Quantitative Biological Methods laboratory. This lab will allow the individual to apply aseptic techniques, use micropipettes, spetrophotometers and the centrifuge. The data that the individual will collect will be used in an analysis to determine the consistency of pipetting. Principle of Methods: This laboratory involved four colored liquid solutions. Each solution was pipetted according to protocol into sixteen 1.5 ml microcentrifuge tubes. After the mixtures were combined and properly capped, each mixture is placed into the centrifuge for several seconds. Once the centrifuge completed its cycle, the mixtures are pipetted into individual cuvettes. Each cuvette is placed in the spectrophotometer and set to 595 nm. After the spectrophotometer calculates each cuvette’s absorbance level, the data is recorded for later examination. Each step in the laboratory was performed using aseptic techniques. A micropipette is a handheld tool that an individual uses to measure and dispense microliters of liquid (Lab Manual). There are multiple types of micropipettes such as a Denville and Eppendorf. Each pipette is different in a few ways. For example, an Eppendorf micropipette uses four digits as opposed to the Denville that uses three digits. An individual may change the volume settings of the micropipette by twisting the knob or plunger. The Denville micropipette displays the volume range on top of the plunger. The Eppendorf micropipette states the volume range on the front and the top near the plunger. While using the micropipette, the correct size micropipette tip is essential for accurate aspiration. The pipette tips are as follows; P2, P10, P20, P200, P1000. To remove a used pipette tip, depress the ejector and discard the tip. Micropipettes should never be turned past the maximum volume as it could damage the tool. The pipette should only be a few millimeters away from the liquid when aspirating; the solution should be dispensed slowly to the first stop to avoid errors and spills then depressed completely to the second stop for total expression. When dispensing and aspirating, ensure the micropipette is directly up and down and the tube about 45 degrees to avoid any issues with gravity, aspirating into the shaft, air bubbles, or a reduction in the accuracy. When dispensing a small amount of a more viscous solution, like solution D in this lab, pipetting up-anddown is a useful technique where the individual depresses plunger several times to the first stop. This technique can be used to mix the contents of the tube or using a vortexer which will vibrate the contents together. In this lab, I did not use the vortexer and instead inverted the contents of the tube before placing them into the centrifuge. The centrifuge is a mechanical device that spins microcentrifuge tubes and their contents at high speeds, separating the liquid and sediment creating a pellet. The centrifuge can be dangerous and the individual placing the tubes must be careful.

The tubes must be placed in such a way that the centrifuge is balanced. A good way to accomplish this is to leave blank spaces across from one another as well as keep samples of equal mass across from each other. If there is only a single space left blank, another tube should be placed of equal mass to the across tube to properly balance the centrifuge. Another microcentrifuge tube layout is triangular if the vectors cancel out. There are many types of centrifuges and rotors with buttons or knobs to set the speed or gravitational force, RPM and RFC, respectively. The spectrophotometer is a laboratory device that measure transmittance and absorbance of light. Transmittance is the amount of light that can be shown through the sample and this is represented as a percentage. The absorbance is the amount of light the sample absorbs and can be used to see the concentration of a sample. Transmittance and absorbance have an inverse relationship and an individual can convert between the two using Beer’s Law. This device has two different lamps that allows for the separation of light to see a particular wavelength. The spectrophotometer uses cuvettes that fit in a certain way and are labeled by a mark or indent. A blank sample is initially inserted to zero out the machine then, the samples are placed into the machine to determine the absorption. Be careful not to zero the machine more than one time, this can give unreliable results. By comparing the transmitted light (I) with the incident light (I 0) the spectrophotometer can calculate the absorbance (Lab Manual). In this lab the spectrophotometer was set to 595 nm.

Materials: •

Solution A

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Solution B

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Solution C

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Solution D

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Micropipettes: Eppendorf Research Plus, Denville XL3000i

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Microcentrifuge: Eppendorf 5418

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Spectrophotometer: Bio-Rad SmartSpec Plus

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Vortexer: Bio-Rad BR-2000 (not used)

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Kimi Wipes

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Gloves

Results:

Discussion: The purpose of this laboratory was to familiarize myself with the techniques and equipment used in the QBM lab by micropipetting, using the sectophotometer and centrifuge. The equation for my data is y = .00552x-.2022, with an R 2 of .556. The value indicates that there has been an error due to the R 2 value being less than 0.95 (Lab Manual). The results were darker than expected and tube number 9 (6ul) deviates from the data the most which can be partially the cause of the R2 value being so low. Unfortunately I used the incorrect Eppndorf micropipette while administering solution D and added more than I was supposed to. When comparing my absorbance values with table 1-3 and 1-4 my values came out significantly higher. I did have somewhat of a trend in my data which Based on the low R2 the labratory was not a success. However, I can conclude that I succeeded in fa...