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 performed in
a 0.2 ml microcentrifuge tube. We did not mix everything together by ourselves. We obtained 47.5 microliter of PCR master, which contains all the other components of the PCR recipe, and then we add it to our 2.5 microliter of DNA sample.
We kept our sample on ice until we were ready to load them into the thermal cycler. After putting the PCR reaction into the PCR machine, the next process was to do the electrophoresis of the PCR product. Our instructor completed that part of the experiment. She obtained two new tubes, and labeled one “DNA” and the other one “water”. She added 20 microliter of the PCR sample and 2.5 microliter of gel loading dye to each tube. The instructor loaded these samples onto a 1% agarose gel for electrophoresis. She loaded the DNA ladder in one of the lanes of the gel. She took a picture of the gel when the blue dye has run halfway down the gel.
Ligation of PCR product into a plasmid and transformation of E. coli
We prepared a 6 microliter of ligation reaction in a labeled 0.5 ml tube by mixing 4.0 microliter of PCR product, 1.0 microliter of salt solution, and 1.0 microliter of vector (plasmid containing topoisomerase I). We incubated on the bench for 10 minutes. During that time, we thawed out top 10 cells on ice, and then put them on a labeled vial of cells. After that, we proceeded to the transformation reaction. We add 6 ml of ligation reaction to our tube of cells and we gently mixed. We incubated on ice for 5 to 10 minutes. We used a machine to heat shock the cells for 30 seconds at 42 C, then we put the cells back into ice for a least one-minute. 250 microliter of warm SOC medium was added to each vial. We shook them horizontally for 1 hour at 37 C. Finally, we used sterile spreaders and spread 50 microliter on the LB plate and 50 microliter on the LB+Amp plate.
Plasmid DNA Minipreps
We retrieved our 2 overnight liquid cultures from our TA. We transferred 1.5 microliter from each culture tube into a 1.5 microliter microfuge tube, and we labeled them E1 and E2. We centrifuged for 1 minute, then we pour out the supernatant into a beaker. We used the vortex machine in order to suspend pelleted bacterial cells in a 250 microliter Buffer P1. We added 250 microliter Buffer P2 and gently inverted the tube 4 to 6 times. We added 350 microliter Buffer N3 and mixed gently for 4 to 6 minutes until the solution became colorless. We centrifuged for 10 minutes at maximum speed. At that time we observed a white pellet. We used a pipet to get the supernatant from each tubes that we labeled QIAprep spin columns. We centrifuged for one minute at maximum speed. We washed the QIAprep spin columns by adding 750 microliter Buffer PE, then centrifuged at maximum speed for one minute.
We discarded the flow-through, and centrifuged for one minute again in order to remove the residual liquid from the bottom. We placed the QIAprep spin column in a clean labeled 1.5 ml microcentrifuge tube. Finally, we added 50 microliter of sterile water to the center of each QIAprep spin column, let it stand for one minute, and centrifuged for one minute at maximum speed. The QIAprep spin column was discarded, and we successfully isolated 50 microliter of plasmid DNA. The instructor did the electrophoresis of the undigested plasmid DNA. She added in this order 16.0 microliter of water, 2.0 microliter of plasmid DNA, and 2.0 microliter of 10X loading dye onto a 1.5 ml tube. She loaded the sample in 1% agarose gel for electrophoresis. She took a picture of the blue dye when it has ran half way down the gel.
Restriction Analysis and Quantification of Plasmid DNA
This experiment was performed in two parts. The first part consisted in determining if DNA has an insert of our PCR fragment. We obtained two tubes of 0.5 microliter, and labeled them. We prepared the restriction digest reaction by adding the following in order: 14.0 microliter of water, 2.0 microliter of 10X EcoRI reaction buffer, 3.0 microliter of miniprep plasmid DNA, and 1.0 microliter of EcoRI enzyme. We incubated the reaction for one-hour minimum at 37 C. We added 2.5 microliter of 10X loading dye to the reactions. All the restriction digest samples were loaded with a 1% agarose gel for electrophoresis. Our TA photographed it. The second part of this experiment was about quantifying our plasmid DNA. It was performed as stated in our lab manual.
PCR Amplification of either the 18S RNA or actin genes.
Lane 5 revealed it was actin
Plasmid DNA Minipreps
At the top in lane 5, is the result of our miniprep.
Restriction Analysis and Quantification of Plasmid DNA
Our purpose in this lab was to determine which gene we cloned between actin and 18S rRNA. From lab 6 results, we considered lane 6, even technically we were supposed to be group five. We cannot clearly see our lane in the picture, so we used lane 5 data. Based on that we can say that we cloned actin. This is one of the most relevant error we had in this experiment, since our lane did not show up properly.
Ahokas, H. and Erkkila, M.J. (1993) Interference of PCR amplification by the polyamines, spermine and spermidine. PCR Methods Appl. 3, 65–8.
Garey, JR, JL Moore, JA Yoder, MC Harmon, and V Carson. 2010. The Genetics Laboratory Manual: PCB3063L. Pro-Copy, Tampa. Print GRUNSTEIN, M., and D. S. HOGNESS, 1975 Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene. Proc. Natl. Acad. Sci. USA 72: 3961-3965. Pierce, Benjamin A. Genetics: A Conceptual Approach. Basingstoke: Palgrave Macmillan, 2011. Print.
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