Biochemistry
9th Edition
ISBN: 9781319114671
Author: Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.
Publisher: W. H. Freeman
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Question
Chapter 5, Problem 12P
Interpretation Introduction
Interpretation:
The affect of altering temperature of hybridization on the PCR amplification is to be stated. The way by which the controlling of stringency of the hybridization helps an individual is to be stated.
Concept introduction:
DNA stands for deoxyribonucleic acid, is a biological macromolecule. DNA contains double helical strands along with the complementary base pairs. The four complementary bases of DNA are adenine (A), thymine (T), guanine (G) and cytosine (C).
The reaction that is used to make the duplicates of a particular DNA segment is known polymerase chain reaction (PCR).
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gene. If the JM109 strain is transformed by the PBKSK plasmid, the strain will produce the B-galactosidase (from the lac gene)
and will hydrolyze X-gal to produce the blue compound. Therefore, colonies that were transformed and contain the pBSKS wil
you
appear blue.
IPTG &
X-Gal &
NO colonies
Amp
E. coli JM109
E. coli JM109
50 mM calcium
chloride-15% glycerol
lac
lac
lac
IPTG &
I Recovery
X-Gal
solution at -702C
PBSKS
White colonies
E. coli JM109
E. coli JM109
ampR
amp I
amp
lac
lac
Heat Shock
Non-transformed
42°C
E. coli JM109
E. coli JM109
amps
amps
lac
lac
IPTG &
X-Gal
lac I
Recovery
lac
PBSKS
BLUE colonies
PBSKS
ampRI
(amp
Transformed
IPTG &
X-Gal &
BLUE colonies
Amp
Hypotheses: Circle the correct answer
1. If PBSKS is transformed into JM109 cells, colonies will be (able/not able) to grow in the presence of ampicillin.
a. Why?
_
2. If PBSKS is transformed into JM109 cells, colonies in media with IPTG (will/will not) induce the production the B-
galactosidase enzyme.
a. Why?_
3. If…
organisms ha
varieties. Through this process, several genetically (10)
been produced.
What I Can Do
Activity 7: MATCHY MATCHY
Direction: Match the purpose to the components found in the box below.
Antibiotic
Resistance Gene
Multiple Cloning Site
Promoter
DNA
Inserted Gene Sequence
Multiple Cloning Site
1. Allows the controlled expression of the desired gene in the
presence of an inducing agent (e.g., beta-galactosidase; heat
treatment (~65°"C)
2. DNA sequence or portion for the insertion of the desired gene.
This section may contain sequences that will be cut by
specific restriction endonucleases ( cuts within the molecule)
3. Successful insertion of a gene allows the expression of its
protein product. This usually provides a specific trait to the
"transformed" bacteria.
4. Provides a way to screen a population of bacteria for those
that took up the plasmid. For example, if an ampicillin
resistance gene is encoded in the plasmid, then only bacteria
10
t4
Pick a plasmid . What was its approximate transformation? Express it in # colonies per microgram of DNA transformed.
Assume the original DNA was about .001 ug/ul . Count how many colonies you got on one plate (or estimate that number) and figure out how much of the total solution you plated on that plate. Multiply by all the plates, if you plated all of it. OR, if you only plated some of it, figure out how many colonies you would have gotten had you plated all of it. Divide by the number of ug used.
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