MIDTERM POWER POINT OUTLINE
I. Power point will be on the Waksman research
II. You can have pictures, diagrams, key words, or outline on power point but no
sentences. You should be able to present without reading from power point.
III. The following should be the outline for presentation:
You should be able to go through all the steps starting with a cell making mRNA to
making a cDNA library to analyzing the gel to DNA sequencing. For each step you
should have a basic understanding of how it is performed, why is it performed, basic
biological principles applicable to step, and interpreting results. The focus should be on
following the insert at every step. How did the insert get positioned there, why was it
positioned there, how can you determine if the insert is positioned there? A guide to help
you is below:
1. What question or problem are you trying to answer in the Waksman project?
2. Why are you interested in obtaining this knowledge?
3. Why use Duckweed?
a. Discuss model organism
b. Explain why information gained from model organisms can be applied
to humans.
c. Give molecular evidence for unity among all organisms.
d. What unique features does duckweed have that make it interesting
for scientists to understand?
4. What are libraries?
a. Describe the two types of libraries
b. What are the advantages and disadvantages of each?
c. What type of library are you using and why?
d. Give a basic description on how to create a cDNA library starting
with a cell.
e. Specifically describe how Waksman synthesized cDNA starting with
mRNA and how it was digested to be inserted into a plasmid. Why the
beginning of insert starts after Scifi A and run of G’s
f. Describe what you did with the library and why?
5. What is an overnight?
a. Basic procedure
b. What is a colony and how did you choose a colony?
c. Purpose of overnight?
d. Why is ampicillin in the LB?
e. Which color colony do you pick & why?
f. How do you know if overnight is poor?
g. What will be bad if overnight is bad?
6. What are vectors?
a. Describe vectors
1
b. Why are vectors important in studying DNA sequences and why do we
put our insert into a vector?
c. Describe how vectors enter into host cells?
d. How do vectors replicate inside host cell?
e. What type of vector (general) was used in this project?
f. What is the specific name of the vector used in this project?
g. Explain important features of our vector (ori, color screen, multiple
cloning site, & selectable marker
h. How was the insert placed in the vector?
7. Purification of plasmid from E. coli bacteria
a. What procedure is used?
b. Explain the basic steps.
c. What happens if overnight is poor?
8. Digest
a. What are restriction enzymes?
b. What is their natural function?
c. What enzymes were used to construct our cDNA library?
d. What enzyme was used to digest insert from plasmid?
e. Explain the purpose of the digest
f. What are the four ingredients of a digest?
g. What order do you add ingredients and why?
h. What is the value of using a reaction mix?
k. After you mix the ingredients of a digest, what do you do next?
l. How much larger will the digest be than the actual insert? Why?
m. How many bands will you expect to see on a gel?
n. What might give you extra bands?
o. What is a partial digest, doublet, PVUII site in insert
9. PCR
a. What is purpose of PCR?
b. What does PCR stand for?
c. Explain how PCR works?
d. What is name of machine that performs PCR?
e. What are the primers?
f. What is contained in the PCR bead?
g. What is Taq-polymerase and why is it unique?
h. How much larger will the PCR band be than the actual size of insert?
Why?
I. How many bands will you expect to see on a gel? Why?
J. Why do you dilute the DNA before performing PCR?
10. Gel electrophoresis
a. What is purpose of gel?
b. DNA is separated based on what two properties?
c. Describe structure of DNA
 What makes DNA negative?
 Where does ethidium bromide attach?
d. What does a band on a gel represent?
2
e. What allows for visualization of DNA under UV light?
f. Explain how you determine the size of insert?
 Digest
 PCR
g. What is the normal size of the plasmid?
 Why is it important to know size of plasmid?
 What is size of plasmid after PCR?
 Explain why
h. What is a ladder?
i. What is the purpose of loading dye?
j. Describe the steps in analyzing a gel? (Include open circle, linear, and
super coiled)
K. What four items do you run on a gel?
L. Which direction does DNA move through a gel and why?
M. What happens if you don’t completely melt the gel in microwave?
N. Changing the percent of the gel does what?
11. Analyzing a gel
a. Insert vs. plasmid
b. How do you know which band is plasmid?
c. Compare and contrast PCR gel and digest gel
d. How do you size?
e. How do you know if gel ran well?
f. What are the three bands in uncut lane of digest?
g. What are a doublet, partial digest, and PVU II site in insert?
h. What is a contamination?
12. DNA sequencing
a. Describe the Sanger (chain termination or dideoxy) method of
sequencing DNA?
b. What primers were used for sequencing?
13. Some additional knowledge to demonstrate that you just love this stuff “A”
student
3