Download AP Biology: Evolution

(Copy the title, problem, and background information neatly in your lab
notebook)
AP Biology: Genetics and Information Transfer
______Big Idea 3
Investigation 9: Biotechnology: Restriction Enzyme Analysis of DNA
Lab Objective:
Digest lambda DNA with 4 different enzymes; analyze the digestion results by gel
electrophoresis on agarose gels.
(Remember to write full sentence answers that incorporate part of the question
for it to make sense or write the full question and answer (sentences). Also write
the headings for each section in your lab notebook.)
Background: Restriction Enzymes and Applications:
Where were restriction enzymes first discovered? (What type of organism?) Why
did this type of organism develop these enzymes?
1. In the RE lab we used lambda DNA which is a “phage”. What does that
mean? Sketch a picture of how it operates. What is lambda DNA commonly
used for?
2. What temperature do restriction enzymes usually work at and why?
3. What is a palindrome? Give an example on a strand of DNA. How does it
apply to biotechnology?
4. List and describe thoroughly at least two uses/applications of restriction
enzymes.
Background: Micropipettes/Electrophoresis:
1. Describe what a micropipette is and what they are used for. Specifically
discuss the volumes they transfer and the difference between mcg and mg.
2. Describe what metrology is and why it is important.
3. Describe the steps you would take to transfer 1 micro liter of enzyme using a
p20 micropipette.
4. Describe what agarose is and what it is used for in the lab setting. Distinguish
between varying percentages of agarose and their uses. Include in your
discussion the purpose of combs.
5. Describe why DNA is negatively charged.
Electrophoresis Background (cont.)
(Copy the following paragraph in your lab notebook and then fill in the blanks)
6. Electrophoresis is used to separate the DNA fragments. DNA is
_________________ charged and moves, under the force of an electric current,
through the matrix of the __________________, toward the ____________
electrode. Molecules separate by __________________, with the
______________ones moving more rapidly through the gel than the
_________________ ones. Buffer is used in the chamber to
_____________________________________________________.
7. List and describe thoroughly at least two uses/applications of gel
electrophoresis.
(Copy the big idea, enduring understandings and learning objective codes in your
lab notebook)
Big Idea 3: Living systems store, retrieve, transmit and respond to information
essential to life processes.
Enduring Understandings
 1C3: Populations of organisms continue to evolve.
 3A1: DNA, and in some cases RNA, is the primary source of heritable
information.
 3A3: The chromosomal basis of inheritance provides an understanding of
the pattern of passage (transmission) of genes from parent to offspring.
Learning Objectives:
 LO 3.5 The student can justify the claim that humans manipulate heritable
information by identifying at least two commonly used technologies. (3A1
& SP 6.4)
 LO 3.13 The student is able to pose questions about ethical, social or
medical issues surrounding human genetic disorders. (SP3.1)
(Copy the procedures heading in your lab notebook and attach the packet)
Procedures: (See Attached)
(Copy the analyzing results heading in your lab notebook and answer the
following questions)
Analyzing Results:
1. Give a brief overview of the purpose of the lab.
2. What was the negative control for the experiment? What did this sample
demonstrate?
3. What was the molecular weight marker/ladder? What was it used for?
4. Fill in the sentence:
My restriction enzyme was __________. It has ________ restriction sites on
lambda DNA yielding _____ DNA fragments. On a 0.8% agarose gel,
______of these bands resolved.
5. Sketch a picture of your gel
6. Title the gel picture RE Digest 3/18/13
 Label and number each lane.
 Below the gel picture or sketch, list and number each lane and describe the
results.
For the enzyme lanes: Describe whether the enzyme worked? Did all the bands
resolve on the gel as it was supposed to according to the RE map? If not
discuss what might have gone wrong and suggestions to solve these
errors in the future.
(Copy the Promega Corporation and Barbara’s tips headings in your lab notebook
and answer the following questions)
Promega Corporation:
1. Describe what types of products the company manufactures and how the
products are maintained and shipped. (Hint: ID kits, RE, experiments in a
box, etc.)
2. Describe at least three jobs/careers that a person could do at Promega. List
several “perks” for employees and why the company provides them. Could
you picture yourself working for a company like this some day? If so, doing
what type of work.
3. Discuss the most interesting part of the trip (do not say lunch!)
4. Do you suggest I take future classes to BTCI for this program? Why or why
not?
Barbara’s Tips for College Bound Science Students:
List, describe and explain as many as you can remember… I will get you
started…(you will need at least 4)
1. READ and diagram the lab protocol before the lab…
(Copy the following heading in your lab notebook, read the information and
summarize the information discussed, then cut out the Figure 5 and Table 1 and
staple into your lab notebook with your graph)
Analyzing Electrophoresis Results (Skill): Graphing a Standard Curve
and Calculating the Sizes of Restriction Fragment Length
Polymorphisms
Mathematical formulas have been developed for describing the relationship
between the molecular weight of a DNA fragment and its mobility (i.e., how far it
migrates in the gel). In general, DNA fragments, like the ones in your evidence
samples, migrate at rates inversely proportional to the log10 of their molecular
weights. For simplicity’s sake, base pair length (bp) is substituted for molecular
weight when determining the size of DNA fragments. Thus, the size in base pair
length of a DNA fragment can be calculated using the distance the fragment
travels through the gel. To calculate the base pair length, a DNA standard,
composed of DNA fragments of known base pair length, is run on the same gel
as the unknown fragments and is then used to create a standard curve.
The standard curve, in this case a straight line, is created by graphing the
distance each fragment traveled through the gel versus the log10 of its base pair
length.
Creating the Standard Curve
As explained above, base pair (bp) length is substituted for molecular weight.
Note that in plotting the standard curve, calculating the log10 of the base pair
length of each fragment is unnecessary because the base pair size is plotted on
the logarithmic axis of semi-log paper. Examine your stained gel on a light box or
other surface that helps visualize the bands.
•
What observations can you make?
•
What quantitative measurements can you make?
1. Examine the “ideal” or mock gel shown in Figure 5 that includes DNA samples
that have been cut with three restriction enzymes, BamHI, EcoRI, and HindIII, to
produce RFLPs (fragments). Sample D is DNA that has not been cut with
enzyme(s). DNA cut with HindIII provides a set of fragments of known size and
serves as a standard for comparison.
2. Using the ideal gel shown in Figure 5, measure the distance (in cm) that each
fragment migrated from the origin (the well). (Hint: For consistency, measure
from the front end of each well to the front edge of each band, i.e., the edge
farthest from the well.). Enter the measured distances into Table 1. (See * and
** notes below the table for an explanation for why there are only six bands
seen but more fragments.)
3. Plot the standard curve using the data from the DNA sample cut with HindIII.
To do this, your teacher might ask you to graph the data directly using Excel
with distance traveled as the (arithmetic) x-axis and the base pair (bp) length as
the (logarithmic) y-axis. Based on this graph, why must the data be plotted using
the log scale? You might want to plot the data again using semi-log paper.
Connect the data point with a best-fit line. However, you should ignore the
point plotted for the 27,491bp/23,130 doublet. When using 0.8% agarose gel,
these fragments appear as one. Congratulations! Your best-fit line is the
standard curve.
4.Now use the standard curve to calculate the approximate sizes of the EcoRI
and BamHI fragments and fill in your table. With a ruler, how can you use the
standard curve to calculate the sizes of unknown fragments?