DNA cloning refers to the process of making multiple copies of a DNA fragment. For the past weeks we have conducted a set of experiments that allow us to clone a specific gene in drosophila. First we started by the process of DNA extraction, which allowed us to isolate the genomic DNA from D. Melanogaster. This process requires the use of lysis in other to extract the DNA and RNA. After extracting the DNA, we it is important to use PCR amplification in order to amplify the DNA template to produce a specific DNA fragment. Another important step in DNA cloning is plasmid isolation. Plasmid isolation allows us to extract a plasmid from a bacterial cell (E.coli). In our experiments, we had to amplify either the 18S rRNA or the actin gene found in D. Melanogaster.
Actin is a major contractile protein found in all eukaryotic cells, accounting for 1-2% of the total cellular protein. As the major component of thin filaments, actin is one of the primary proteins responsible for muscle contraction. This protein is also found in D. Melanogaster. 18S rRNA genes constituent of the 40S subunit of eukaryotic ribosomes. 18S rRNA is involved in the initiation of polypeptide synthesis.
After conducting this experiment, at the end we should be able to determine which gene that we cloned from D. Melanogaster: 18S rRNA actin.
PCR Amplification of either the 18S RNA or actin genes
1In this experiment, we used the diluted genomic DNA stock that we had prepared on the previous in order to amplify a portion of either the 18S RNA or actin genes by using PCR. To begin, we had to prepare our 50.0 microliter of PCR composed of 2.5 microliter of DNA template, 37.0 microliter of water, 5.0 microliter of 10X Taq Polymerase Buffer, 4.0 microliter of 2.5M dNTP mix, 0.5 microliter of 20 mm Forward primer, 0.5 microliter of 20 microliter Reverse primer, and 0.5 microliter Taq Polymerase. All that was...
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