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Subcloning is a technique used to produce recombinant DNA. A fragment of DNA containing a gene of interest is inserted into a vector/ plasmid DNA that replicates independently of chromosomal DNA to produce recombined DNA. In order for DNA to recombine, isolation, purification, quantification, digestion, electrophoresis, ligation, transformation, and screening must for all intents and purposes be performed. First alkaline lysis is used to isolate the vector and insert of choice, from bacterial cultures, by opening up the bacterial cell wall and releasing plasmid DNA. Purification removes RNA and protein that may contaminate insert and vector DNA. Quantification reveals the amount of DNA that was isolated (Schramm, Molecular & Cellular Biology Laboratory.). Digesting the DNA sequences with restriction enzymes allows for the extraction of insert and vector DNA at proper sites, which will determine the purity of the DNA samples by using agarose gel electrophoresis (Schramm, Molecular & Cellular Biology Laboratory.). Ligation of the insert to the vector is performed and then transformed into competent cells and grown on plates containing a selectable marker. To determine if the preceding procedures mentioned were successful, the DNA is isolated by doing a restriction digest and the recombinant gene is screened (Schramm, Molecular & Cellular Biology Laboratory). In this particular experiment, if the procedures are carried out successfully the vector will take in the insert gene containing ampicillin resistance and as a result bacterial growth should be seen in the presence of ampicillin and isolation of this recombinant DNA should be possible.
Isolation and purification of DNA from a bacterial culture of E. coli with a vector/plasmid DNA sequence from another bacterial culture of E. coli containing ampicillin resistance, as an insert. An alkaline lysis, with SDS detergent, was used to isolate the DNA from the E. coli cells along with COLD lysis solution. The lysate was incubated at room temperature for 3 minutes and then poured into a spin column and centrifuged, then washed with wash buffer. The DNA was eluted with water, and then underwent centrifugation twice while decanting the flow through, which was then used for quantification while the spin column was discarded. Two vectors and two inserts were used to increase the chances of obtaining purified vector and insert. For quantification, 5 tubes were used, 1 blank (water), 2 separate tubes each containing inserts, and 2 separate tubes containing vector. The blank contained 1000ul of water, each of the 4 tubes contained 5ul of vector DNA and insert DNA and 995ul of water. A spectrophotometer was used to determine the concentration of DNA, and the absorbance. Calculation of the volume of vector and insert was done to obtain 5ug of DNA to be digested by restriction endonucleases Xba1 and BamHI.
Subsequently, 1% agarose gel was prepared with dissolved TAE buffer and ethidium bromide and then solidified. The vector and insert DNA was loaded into the gel. Electrophoresis was done by running the gel for an hour at 120V. Vector1 and insert1 were excised out of the gel as they were highest in concentration of DNA, and then weighed. Three volumes of binding buffer was added to every volume of gel slice and incubated at 50oC for 15 minutes. The original volume of vector and insert of isopropanol was added and mixed by inversion to the DNA samples. Wash buffer was put in and then centrifuged and the filtrate were removed. Centrifugation, elution with 20ul of water, and another centrifugation were done. Next, ligation of the insert to the vector was performed by joining 100ng of vector with no insert (a1:0 ratio), with an equal molar concentration of insert (a 1:1 ratio), and then 3 times the concentration of insert with the vector (a 1:3 ratio). The molar ratio of insert to vector was determined to calculate the volume of the vector and inserts. These volumes were adjusted to 10ul of deionized water. Ligation buffer was added and then mixed. T4 DNA ligase was added and then mixed and centrifuged briefly, then incubated at room temperature for 15 minutes. Later, 5ul of the ligation mixture was transferred into a microfuge tube. Competent cells were added to the DNA and gently mixed by pipeting up and down. The mixture was incubated on ice for 30 minutes and heat shocked for 2 minutes at 37oC, and then cooled on ice for 5 minutes. Luria broth was added, followed by incubation of the cells for 1 hour, 37oC. These mixtures were added to and spread on the appropriate plates. The plates were then incubated at 37oC overnight.
Screening and purification of the recombinant DNA were carried out. Bacterial culture was pelleted, followed by removal of supernatant. This step was repeated with COLD lysis buffer, followed by constant vortexing. The lysate was incubated at room temperature for 3 minutes, and then centrifuged. Wash buffer was added to the tube, followed by centrifugation, removal of the filtrate, decant, and then centrifuged again. The sample was eluted with 30ul of water and centrifuged. Then, DNA quantification was performed. Restriction digest, with the enzyme HindIII, of the quantified recombinant DNA, with a master mix of BSA, Restriction buffer enzyme buffer, and restriction enzyme A, in one microfuge tube was performed. Four more tubes were filled with 3ul of DNA each, and 4ul of H2O, and 3ul of master mix into each of the 4 tubes. These tubes were spun for 1 minute, and incubated at 37oC for 30 minutes. Lastly, 2ul of 5xdyes was added to each tube, so they can be used in electrophoresis, in 1% agarose gel.