Once you have idenified a gene that you suspect is responsible for the disease, you clone the gene in a vector and grow it in the lab (outside the alligator). Which assay would allow you to modify the DNA of the gene in order to figure out what the gene does? O Northern Blot Southern Blot site specific mutageneis O pull down assay / CHIP assay O Differential Display Expression Vector Assay O SAGE O Gene Gun
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- i have a gene (3222bp) i want to put it into a expression vector (pet 28b+),when i did pcr i flanked my insert with EcoRI and HindIII restriction sites,how do i ensure that i insert the gene in the correct orientation in the vector,so that translation of the gene into a protein occurs.if the gene is inserted in the wrong direction would that mean i cant translate or would it just give me the wrong protein produced. also is the vector the one providing the start and stop codon ,and if so how do insert my gene so that it has a start and stop condon in the right framOne concern about using genetically-modified organisms is that many of the methods used to create them introduce into the genome DNA from a different species (i.e. foreign DNA). Which of the following methods has the lowest potential of introducing foreign DNA into the genome?A. A gene knockout in mouse using homologous recombination in ES cellsB. Introduction of a P element vector into the Drosophila germ-lineC. Deletion mutations introduced by CRISPR/Cas9D. Microinjection of a transgene into a mouse pronucleusThere may be more than one correct answer, select all that apply. To clone genes, a plasmid vector should contain a(n) a.) telomeres b.) orgin of replication c.) antibiotic resistance gene d.) restriction endonuclease cut site The CRISPR/Cas system has revolutionized the efficiency at which scientists can experimentally induce a mutation in a gene to investigate the functional role of the protein it encodes. Which of the following are required in the CRISPR/Cas system? Cas9 nuclease Bacterial genome Access to target gene Guide RNA complementary to gene of interest
- You are interested in synthesizing a variant of Synechocystis sp. pcc 7803 ferredoxin having mutation (E) at its 38th residue. The gene is encoded in a pET21b vector (5.3 kbp). Estimate the amount of time needed to run a PCR for the site-directed mutagenesis for25 vs 30 cycles.Site directed mutagenesis is used to: 1.determine the critical base per sequences in a Genome 2. Create genomic libraries 3. Determine the critical amino acids in a protein that allowed to function 4. Amplify dna 5. sequence dnaA more modern molecular technique to RFLP fingerprinting is called Amplified Fragment Length Polymorphisms (AFLPs). In AFLP analysis, restriction enzymes are again used to digest genomic DNA into multiple fragments. Next, adapters complementary to restriction site overhangs are ligated to the fragments using an enzyme called DNA ligase. These adapters are complementary to primers used to amplify the fragments using the polymerase chain reaction (PCR). Can you think of any potential benefits of AFLP analysis over RFLP? Explain your reasoning.
- Probes for cloned genes use ____. a. certain bacteria that glow when they take up the genes b. specific antibodies that kill all the cells c. complementary nucleotide sequences tagged with a detectable label d. specific enzymes that lyse all the cells e. DNA ligaseAfter transduction we move to mutagenesis analysis. Using the transduction plate,PCR I found that lacZ,fliE,nagC,ompA all show successful transduction and successful PCR. But for DNA sequencing some clonies are not correct. Expected size of PCR are lacZ 304,fliE 255,rpoA 420,nagC 329,ompA 731,lexA 350,kan cassette 549,tet cassette 265. Please indicate which mutation were successful and success why some were not from the size about. indicate at what stage any of the mutagenesis failed.You are fresh recruits to a molecular biology laboratory, and your new boss has tasked you tostudy 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 differentsamples through PCR. You will need to run a total of 7 PCRs: 3 normal samples, 3 canceroussamples, and 1 negative control. To make things easier in the lab, when running multiplereactions, the components are prepared not individually, but as a master mix—all thecomponents for multiple reactions are prepared in bulk, except for the template DNA, which isadded 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 thesecomponents, and the needed working concentrations for the PCR itself. Complete the table bysupplying the needed volumes of each component for a single…
- As a part of undergrad lab project, you have to clone Your Favorite Gene into plasmid cloning vector (4740 bp) pUC18. Restriction maps for both YFG and cloning vector are provided. The plasmid has a bacterial origin of replication (ori), an ampicillin resistance gene (AmpR) that degrades ampicillin antibiotic. It also has LacZ gene that encodes the β-gal enzyme, which converts white X-gal substrate into a blue product. pLac is a bacterial promoter. Restriction Enzyme Recognition Site : A slash (/) represents the cut site for each restriction enzyme. Enzyme 1 5’- G/TCGA C -3’ 3’- C AGCT/G-5’ Enzyme 2 5’-C/TCGA G-3’ 3’G AGCT/ C5’ Enzyme 3 5’- GTC / GAC- 3’ 3’- CAG / CTG- 5’ Enzyme 4 5’-C AATT /G-3’ 3’-G/ TTAA C-5’ Enzyme 5 5’C AATT/G-3’ 3’G/TTAA C-5’ Enzyme 6 5’G/GATC C-3’ 3’C CTAG/ G-5’ Enzyme 7 5’G/GATC C-3’ 3’C CTAG/ G-5’ 1a) The table below shows restriction enzyme pairs used by various teams to digest the YFG and clone it in the…You want to clone a specific PCR amplicon. You have determined that the amplicon you want to clone has enzyme restriction sites for HindIII and EcoRI. After investigation you have seen that the pUC18/19 plasmid also have these enzyme restriction sites in its multiple cloning site (MCS on map included). After enzyme digestion your amplicon is 854 bp long. What length will the recombinant plasmid be after you have inserted your amplicon? Show a calculation.Using the techniques below in the laboratory experiments, devise a detailed methodology in solving the situation. -Dna Extraction -Electrophoresis and Quality Checking -Restriction Enzyme Digestion -Polymerase Chain Reaction -Southern Blotting Technique 2. The researchers want to determine if there is a mutation in a carabao farm that leads to high marbling of carabeef marbling. The researchers are knowledgeable and have unlimited funding. In their laboratory they have a PCR, a southern blot chamber and all the materials for DNA extraction. Propose a way to detect if there is a mutation in the carabao marbling.