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).

Stock Working Volume for 1 Volume for 7

PCR Component
concentration concentration reaction (μL) reactions (μL)

Buffer for Taq

10X 1X 1.0 7.0
polymerase

Forward primer 40 uM 1.0 uM

Reverse primer 10 uM 1.0 uM

dNTPs 25 mM 1 mM

Taq polymerase 200 U/ul 1 U/ul

Sterile distilled (to fill reaction

n/a
deionized water volume to 10 ul)

Total 9.0 ul 63.0 ul

1.0 (added separately to each reaction

Template 400 ng/ul 40 ng/ul after master mix is distributed to
different tubes)

Total 10.0 70.0

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.

1. Determine the 5'-to-3' sequence of the wild-type TEMPLATE strand.

Answer: 5’ ___ ___________ ____ 3’
2. Using the codon table given below, determine the corresponding wild-type and mutant amino
acid sequences in order to determine the mutation that occurred. Assume that the sequencing
template is the CODING strand for the NRAS gene. (Use the 3-letter abbreviations for the amino
acids.)

Wild-type amino acid sequence: _________________

Mutant amino acid sequence: _________________
Mutation: The original amino acid __________ was mutated into ________
Bonus : In your PCR master mix, you used Taq polymerase. What species is this enzyme derived
from? Answer: _____________________