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Molecular Strategies And Techniques Of GAPC

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The following project outlines the laboratory portion of associate degree undergrad genetic science course. The course stressed the understanding of molecular strategies and techniques through isolating, cloning, and expressing a sequence referred to as GAPC (a gene). GAPC has denoted the sequence that codes for cytosolic GAPDH. GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) is a catalyst that chemical process for a metabolic process (which is glycolysis (stage of respiration that happens all toldliving cells)) from an uncharacterized plant species. GAPDH is additionally thought of a gene due to being referred to as a housekeeping gene that continuously is expressed and is involved in basic cell functions. The importance of GAPDH is that the sequence is found within the order of all plants, moreover, as all told alternative organisms. GAPC is that the main gene that is being sorted through the full experiment. For the cloning and sequence project, the two species that would be looked upon for the semester were Acer Palmatum (Japanese Maple) and Acer x freemanii ‘Sienna’ (Sienna Glen Maple). These two plants contain a specific gene called GAPC which is derived from GAPDH.

For the gene family, GAPDH is the original gene that was duplicated to make GAPA, GAPB, and GAPC/ GAPCP. GAPC is found in Arabidopsis thaliana (flower plant of the mustard family) which has been experimented on to help with further research on the extended line in GAPDH in plants. For, the cellular function of GAPC is a gene that helps codes a conserved cytosolic enzyme that helps in glycolysis (is the process in which one glucose molecule is broken down to form two molecules of pyruvic acid) and catalyzes other enzymes. (Rius, Casati, Iglesias, and Gomez-Casati, 2008). The purpose of the experiment was an attempt to clone GAPC and sequence it from Japanese Maple and Sienna Glen Maple. The hypothesis of this experiment is the sequences of the genes from Japanese Maple and Sienna Glen Maple will be similar to each other based on their genetic coding that is distinct; The reason behind this is that all plants have GAPDH with a different variation. Since GAPA, GAPB, and GAPC/ GAPCP originated from GAPDH it makes sense that there will be a similarity between the genes. This experiment was performed, by having Japanese Maple and Sienna Glen Maple plant materials grounded in separate tubes and centrifuged. Then the DNAs were purified by using a silica spin column.

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After that, the DNAs were eluted from the spin column (DNA extraction step). A master mix was added to the samples and positive controls, amplified, and treated with exonuclease I to remove primers for the initial PCR. Then nested PCR, master mix, and PCR primers were added to the initial PCR samples and positive controls DNAs. After that, they were amplified and analyzed with DNA electrophoresis (Two PCR steps and electrophoresis step). . Purified and blunted PCR products were combined with a pJet plasmid vector, and T4 ligase and ligated for 10 minutes and then transformed into bacteria and incubated overnight (Ligation and transformation step). Liquid bacteria were grown in a culture overnight (shaken water bath) and restriction digest with purified Plasmid DNA using alkaline lysis was placed in miniprep spin columns (Restriction Enzyme analysis and electrophoresis step). Lastly, combined performed plasmid contained cloned geneswith sequencing primers were mailed to sequencing faculty and analyzed using Geospiza and web portal (Sequence and bio-information step).


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