= Menu #2 Mol Bio Mutation QU... X + Create All tools Edit Convert E-Sign Q Search Complete the following tasks. Sign in Find text or tools Q H 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 Stock concentration Working concentration Volume for 1 reaction (µL) Buffer for Taq polymerase Forward primer Reverse primer dNTPs 10X 1x 1.0 40 uM 1.0 uM 10 uM 1.0 UM 25 mM 1 mM Tag polymerase Sterile distilled deionized water 200 U/ul 1 U/ul n/a (to fill reaction volume to 10 ul) Total 9.0 ul Template 400 ng/ul 40 ng/ul S Total Volume for 7 reactions (μL) 7.0 63.0 ul 1.0 (added separately to each reaction after master mix is distributed to different tubes) 10.0 IA W 70.0 ☑ 0 Al Assistant 1 3 C ༢ ☑ 8:54 PM 10/23/2024 1:1 Q = Menu #2 Mol Bio Mutation QU... X + Create All tools Edit Convert E-Sign Sign in Find text or tools H Q 2 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 MUTANT NRAS ddA ddT ddC ddG ddA ddT ddC ddG 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.) Second LUC QUA LUG CUM He CLA CCA CM CA CUG AUT AQU ACC AUA ACA AGA A GLC GCA Q Search Wild-type amino acid sequence: Mutant amino acid sequence: Mutation: The original amino acid was mutated into IA Σ ☑ ༢ ☑ 0 Al Assistant C Dr 2 3 C 1:1 8:54 PM 10/23/2024 Q