Complete the following tasks. You are fresh recruits to a molecular biology laboratory, and your new boss has tasked you to study mutations in NRAS, a gene that she suspects is involved in cancer pathogenesis. Task A: Polymerase Chain Reaction Master Mix Your first task is to isolate and amplify the NRAS gene from cDNA extracted from different samples through PCR. You will need to run a total of 7 PCRs: 3 normal samples, 3 cancerous samples, and 1 negative control. To make things easier in the lab, when running, multiple reactions, the components are prepared not individually, but as a master mix-all the components for multiple reactions are prepared in bulk, except for the template DNA, which is added separately once the master mix has been distributed into individual tubes. The table below lists the different components for PCR, the available stock concentrations of these components, and the needed working concentrations for the PCR itself. Complete the table by supplying the needed volumes of each component for a single reaction and for the master mix (the first row has been filled in as an example). PCR Component Buffer for Taq polymerase Forward primer Reverse primer dNTPs Taq polymerase Sterile distilled deionized water Template Stock concentration 10X 40 uM 10 uM 25 mM 200 U/ul n/a Total 400 ng/ul Total Working concentration 1X 1.0 uM 1.0 um 1 mM 1 U/ul (to fill reaction volume to 10 ul) 40 ng/ul Volume for 1 reaction (µl.) 1.0 Volume for 7 reactions (μl) 10.0 7.0 9.0 ul 63.0 ul 1.0 (added separately to each reaction after master mix is distributed to different tubes) 70.0

Human Anatomy & Physiology (11th Edition)

11th Edition

ISBN:9780134580999

Author:Elaine N. Marieb, Katja N. Hoehn

Publisher:Elaine N. Marieb, Katja N. Hoehn

Chapter1: The Human Body: An Orientation

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Task A: Polymerase Chain Reaction Master Mix Your first task is to isolate and amplify the NRAS gene from cDNA extracted from different samples through PCR. You will need to run a total of 7 PCRs: 3 normal samples, 3 cancerous samples, and 1 negative control. To make things easier in the lab, when running multiple reactions, the components are prepared not individually, but as a master mix—all the components for multiple reactions are prepared in bulk, except for the template DNA, which is added separately once the master mix has been distributed into individual tubes. The table below lists the different components for PCR, the available stock concentrations of these components, and the needed working concentrations for the PCR itself. Complete the table by supplying the needed volumes of each component for a single reaction and for the master mix (the first row has been filled in as an example).

Complete the following tasks.
You are fresh recruits to a molecular biology laboratory, and your new boss has tasked you to
study mutations in NRAS, a gene that she suspects is involved in cancer pathogenesis.
Task A: Polymerase Chain Reaction Master Mix
Your first task is to isolate and amplify the NRAS gene from cDNA extracted from different
samples through PCR. You will need to run a total of 7 PCRs: 3 normal samples, 3 cancerous
samples, and 1 negative control. To make things easier in the lab, when running multiple
reactions, the components are prepared not individually, but as a master mix-all the
components for multiple reactions are prepared in bulk, except for the template DNA, which is
added separately once the master mix has been distributed into individual tubes.
PCR Component
Buffer for Taq
polymerase
Forward primer
Reverse primer
dNTPs
The table below lists the different components for PCR, the available stock concentrations of these
components, and the needed working concentrations for the PCR itself. Complete the table by
supplying the needed volumes of each component for a single reaction and for the master mix
(the first row has been filled in as an example).
Taq polymerase
Sterile distilled
deionized water
Template
Stock
concentration.
10X
40 uM
10 uM
25 mM
200 U/ul
n/a.
Total
400 ng/ul
Total
Working
concentration
1X
1.0 uM
1.0 uM
1 mM
1 U/ul
(to fill reaction
volume to 10 ul)
a
40 ng/ul
e
Comment Highlight Draw
ddA
Volume for 1
reaction (μL)
go
1.0
10.0
Volume for 7
reactions (ul)
9.0 ul
1.0 (added separately to each reaction
after master mix is distributed to
different tubes)
MUTANT NRAS
ddT ddC ddG
7.0
Task B [5 points]: Sanger sequencing
Having successfully amplified the NRAS gene from wild-type and cancerous tissue through PCR,
you now sent your PCR product for sequencing. However, the only available technology to you
is classical Sanger sequencing or the dideoxy chain termination method, explained in Figure 2.
Shown below are the sequencing gels obtained for the sections of the wild-type and mutant NRAS
genes where the mutation can be found.
WILD-TYPE NRAS
ddA ddT ddC ddG
63.0 ul
70.0
T
000
000
000
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