Dna Purification Lab Report

The purpose of module E is to learn several DNA techniques in the lab including DNA purification with solubility and absorption, plasmid transfection of E.coli, colony screening by PCR and quantitative PCR.
First part of the experiment E1 show the purification method of DNA through solubility. E. coli lysate mixed organic solvents to purify the DNA present in solution. First, the lysate was mixed with phenol/chloroform, then vortexed, and centrifuged. We extracted the aqueous layer and combined with sodium acetate and cold ethanol and then centrifuged and filtered. The ethanol causes the DNA to precipitate since the contaminants were removed from the previous precipitation. The remaining pellet was virtually invisible, but, when re-suspended with TE buffer, produced a reading in the UV spec.
Bacteria naturally take in loops of DNA from the environment called plasmids.
These plasmids can be inserted into the bacterium’s genome which may increase their chances of survival through diversity. This method was done in the lab by using vector transfection. Vectors are the plasmids that are inserted into bacterium for protein expression. Vectors are added to solution with bacteria exposed to heat-shock so they take up the DNA. These bacteria are then allowed to grow and replicate on agar included vector. Some indicator is also added the vectors to indicate which colonies have inserted the vectors.
Experiments E2 and E3 were transfection of vectors and analysis of transfection respectively. The vector being used included an ampicillin gene as its selective gene and a fluorescence gene induced with arabinose. Three agar plates were prepared with three different samples. Sample 1 was a control and included only LB, sample 2 included LB and ampicillin, sample 3 included LB, ampicillin, and
arabinose.
Replication of the vector is done via PCR. There are three steps; denaturing, annealing, and elongation. Double-stranded DNA vectors are added to buffer solution with primers, Mg ions, dNTPs, and DNA polymerase (often Taq polymerase). The mixture is heated so the DNA denatures. The mixture is then cooled so the primers anneal to the template strands. Finally, it is heated to the preferred temperature of the polymerase for elongation. The steps are repeated to produce a theoretical yield of 2n copies.
Quantitative PCR is an analysis method that determines the amount of DNA in solution at the onset of the experiment. This is accomplished when fluorescence is added to DNA as it replicates. The PCR equipment reads the amount of fluorescence after each cycle and compares this to a value (Cq). The signal passes Cq after X number of cycles compared to the baseline DNA in the experiment. Centrifugation is a technique used to separate molecules in solution. It takes advantage of simple physics and properties of particles. Organic solvents were used that made lipid and protein contaminates insoluble in solution while DNA remained soluble. These solutions were centrifuged which pushed the insoluble contaminants to the bottom (referred to as the pellet). The supernatant (liquid part remaining) included the DNA to be removed for further experimentation.