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Final

01:119:117 Study Guide - Final Guide: P200, Carbon Fixation, Enzyme


Department
Biological Science
Course Code
01:119:117
Professor
Torress
Study Guide
Final

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Bio Lab Final
Lab 1
P-20 for dispensing 1-20 ul
P-200 for dispensing 20-200 ul
P-1000 for 200-1000 ul
The P-20 and P-200 pipets use the same disposable tip (usually white or yellow)
that attach to the white stem
The P-1000 uses a larger tip (blue).
You can also transfer liquids using a plastic Pasteur transfer pipet
oThese pipets have marked gradations on the side and are used to transfer
liquids that are less than 1 ml.
oThey are less accurate than using the pipetman
The volume indicator consists of three number dials that are read top to bottom
oThe P-20 is calibrated in 0.1 ul increments, the P-200 in 1 ul increments,
and the P-1000 in 10 ul increments
*Never adjust the pipetman above the maximum volume or below the minimum
volume for a particular pipetman
Push the plunger down to the first stop BEFORE entering the liquid you want to
pipette
Percent error (%) = (theoretical mass – measured mass)/theoretical mass x 100
It is important to run experiments repeatedly, in duplicate and triplicate, to ensure
accuracy.
Many experiments require proteins to carry out a particular function, such as
binding to DNA or cleaving a substrate
oThe activity of these proteins is often very dependent on the pH, salt
concentrations, and temperature of the reaction mixture.
Buffer: aqueous solution containing a specific mixture of salts, buffering agents,
and sometimes reducing agents, detergents or cofactors, in which each of the
components has a purpose and is included to optimize the reaction
oExtremely important in helping maintain the pH of a solution
In order to save time and space, biologists often use stock solutions, concentrated
solutions that last over long periods of time
oThese stocks are easily diluted for use when necessary
oUsually diluted with water
This equation is used to make a specific volume of a dilute solution from a stock
solution:
oC1V1=C2V2
Dilution factor : factor by which the concentration of the dilute solution is reduced
compared to the concentration of the stock solution
oDF = C1/C2 = V2/V1
Serial dilution : stepwise dilution where the stock solution for each dilution in the
series is the dilute solution from the previous dilution.

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oThe total dilution factor is the product of the dilution factors for each
dilution step
oDFtotal=DF1 * DF2 * DF3, etc.
Serial dilutions result in a series of solutions that are diluted by a certain
numerical factor and can be used for making standard curves.
SpectroVis : a spectrometer, a machine that can measure absorbance or
transmittance of a pigmented solution
oBiologists commonly use them to quantify the concentration of materials
in a solution
oThis instrument produces a beam of light with a specific wavelength that
passes through the sample before entering a photometer that measures the
amount of light
This measurement is transformed electronically to a reading on a
meter that quantifies the amount of light absorbed
The SpectroVis can shine specific wavelengths of light onto a sample
o“white” light is actually a mixture of different wavelengths
Ex: if you shine white light on a red solution, the light appears to be red because
the wavelengths corresponding to red are reflected and transmitted through the
material.
oThe other wavelengths, such as blue, are ABSORBED.
The amount of light absorbed at different wavelengths is quantified using a
spectrophotomer.
HOW the SpectroVis works:
oThe SpectroVis produces a beam of light with a specific wavelength
(color) that passes through the sample before entering a photometer that
measures the amount of light reaching the photometer.
*The absorbance of a sample is directly proportional to the concentration of
material in the sample
Standards have a known amount of the material being assayed and are used to
calculate the amount of material in the unknown.
Two ways to determine the concentration of an unknown sample:
oUse equation Cs/Cu=As/Au
oMake a standard curve
Standard curve : tool scientists use to determine the unknown
concentration of a sample
oGenerated by measuring the absorbance of a series of standards and
graphing the absorbance as a function of concentration.
oThe unknown concentration of a sample can be determined by
interpolation of the graph.
Blank: sample used to calibrate zero absorbance on the spectrophotometer.
Set the spectrophotometer to zero absorbance using the sample with only
reagents.
oThis corrects for any absorbance due to the reagents and not to the
materials being assayed.

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**The blanks in our procedures include the reagents
Correlation: statistical tool which helps describe the relationship between two
variables and how closely they correspond with each other
R^2 value will tell you how closely your data fits with the linear trendline
Since absorbance is directly related to concentration we should have a perfect R^2
of 1.
If your group gets an R^2 of 0.97 or better you should be fairly confident in your
results.
oIf you get a lower R^2, there are several sources of error
Equipment, pipetting, clean test tubes, calibration as well as the
accuracy of the person completing the dilution
Graphs must include following:
oTitle
oGraph format : the independent variable on the X-axis and the dependent
variable is plotted on the Y-axis.
oLabels on all axes: what variable that each axis represents. Include units
for variables.
Ex: “time” but what unit of time, days, minutes, etc.
oLegend : An explanatory paragraph that reviews the content of the figure.
oKey : figures with more than one set of data only need a key
*A true legend is NOT the same thing as what Excel calls a “legend” when you
construct a graph.
You should always hold and label the spec tubes at the top half of the tube to
avoid fingerprints and marks in the light path that can affect the absorbance
values.
Example question: If a solution is red, does that mean red wavelengths are
absorbed or transmitted by the solution?
Red wavelengths are transmitted by the solution.
After you make your serial dilution, to determine the absorbance of the all the
samples, which wavelength should you use?
oThe range for all colors absorbed except the color of the samples
(including stock solution)
Lab 2
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