Experimental Design #2: Identifying an Unknown Plasmid
1. The purpose was to isolate a plasmid from E. coli and digest it using restriction
enzymes to identify it. Plasmid Identification is useful in that it can be used together with othe
1) What reagent(s) can be used to make cells competent?
Correct: All of the listed choices can be used to make competent cells
2) In order to have an on/off switch
MCB 251- C
Analysis of Unknown Digestion
I think the unknown plasmid that I have received is pAMP because its the only plasmid that
has around 4500 base pairs which is what I think the uncut band size is.
1) Which of the following is not a mechanism to regulate gene expression?
Incorrect: Phosphorylation of proteins
Incorrect: Transcription of DNA
Incorrect: mRNA degradation
Incorrect: Intron removal
Correct: Regulatory mutan
Laboratory Notebook Assignments
All MCB 251 students are required to keep a laboratory notebook. See Required and
Recommended Materials section for the exact make of notebook required. The lab
notebook assignments will be collected the week following the
Identifying an Unknown Insert
MCB 251- C
The purpose of this lab is to determine the unknown insert by using your unknown
plasmid DNA as a vector to clone your unknown insert. You will then transform your bacteria by
having it take in t
PCR Cycle Sketch (20 points)
For this assignment you will be drawing several cycles of PCR and answering a few short questions. The
following animations will help you to understand the processes of PCR and gel electrophoresis.
The Polymerase Chain Reactio
Polymerase Chain Reaction
Add the following to a PCR tube, and in this order
(these look like small Eppendorf tubes)
2X Paq5000 Master Mix
5 mcL Primer P1/Upstream Primer
5 mcL Primer P2/Downstream Primer
2.5 mcL Template
Cloning Final Week
Also the end of IE#3
Running Out the cat PCR Products
To each PCR tube from last week, add 20 L of loading dye.
Mix with a micropipette by pipetting up and down.
Get a 1.0% agarose gel.
Load a ladder lane with 5 L prepare
Phages & Transformation
1. Transform an E. coli strain (DH5 pir) with a plasmid
encoding, among other things, ampicillin resistance
2. Infect two different strains of E. coli with phage to
test for resistance and to demonstrate mutation
Experimental Control Clarification
Controls are used to assure that experimental results are
Positive control: a positive control should give the desired outcome of
the experiment, provided all the reagents and equipment are
Gel electrophoresis: sort and see the DNA
Go to the DNAi website www.dnai.org > Manipulation > Techniques > sorting and sequencing.
2. View the Gel Electrophoresis 2-D animation, and answer the following questions.
Phages & Transformation
1. Determine the nature of the mutations in the E. coli
cells infected last week which confer a R phenotype.
2. Determine mutation frequencies of E. coli w.t. and
1. Determine concentration and numb
Isolation of a Plasmid and
Objectives for the Week
A. Continuation of Independent Exercise (IE) #1
1. Collect data, prepare report for submission next week.
B. Isolate and restrict a plasmid
1. Isolate and prepare a plasmid from a bact
IE#2 Lab Report Analysis of Unknown Digestion
The purpose of this lab is to identify which plasmid was digested and to prove that it is indeed that
plasmid through a calculated digestion paired along with gel electrophoresis to visualiz
Hand Washing Experiment
To see if washing your hands with soap, in addition to water, really does eliminate a significant
number of bacteria from your hands, as opposed to only washing your hands with water.
Washing hands along with s
pBR322 Digestion Lab Analysis
1. Get uncut, and digested pBR322 samples that were cooled on ice. Take five of these
2. Add 10 microliters of 5x blue loading solution to each tube labeled (B, E, P, EP, and C).
3. Flash spin each sample in