E. coli Genomic DNA Isolation and Quantitation
Introduction
In this experiment, the genomic DNA of E. coli cells were isolated, purified, and quantified. To achieve this, E. coli cells were lysed, purified from impurities and
Methods
E. coli cells were first centrifuged in order to separate the cell pellet from the supernatant. Cell pellets were resuspended in saline-EDTA, which weakens bacterial membranes and chelates metal ions. Since E. coli has three layers (outer membrane, peptidoglycan layer, and the inner membrane) saline-EDTA helps break the outer membrane by removing the cations that link the phosphate groups in the ends of lipopolysaccharides. Once the cations are removed, the negative ends of the phosphates repel and pierce holes in the membrane. After the resuspension, lysozyme solution was added, which breaks down the peptidoglycan layer by breaking the glycosidic bonds that hold together NAG and NAM …show more content…
As seen in the table above, none of the determined ratios fall within the range. The fact that none of my A260:A280 ratios fell in the range, and only one of my samples fell in the A260 desired range, indicates that there was significant protein contamination in my sample. This could be due to poor pipetting skills, leading to contact with the interface layer when extracting the aqueous layer. In addition, this could also be due to poor mixing, as proper mixing is essential for SDS and sodium perchlorate to denature proteins and inhibit their contact with DNA.
Since only one of my samples had an A260 values that fell within the desired range, the average concentration cannot be calculated. Using the volume of water used to dissolve DNA, and assuming Sample B represents average concentration, the yield of the isolated genomic DNA can be calculated. Yield = average concentration volume of water used Yield = 405g/mL7mL =
Introduction
In this experiment, the genomic DNA of E. coli cells were isolated, purified, and quantified. To achieve this, E. coli cells were lysed, purified from impurities and
Methods
E. coli cells were first centrifuged in order to separate the cell pellet from the supernatant. Cell pellets were resuspended in saline-EDTA, which weakens bacterial membranes and chelates metal ions. Since E. coli has three layers (outer membrane, peptidoglycan layer, and the inner membrane) saline-EDTA helps break the outer membrane by removing the cations that link the phosphate groups in the ends of lipopolysaccharides. Once the cations are removed, the negative ends of the phosphates repel and pierce holes in the membrane. After the resuspension, lysozyme solution was added, which breaks down the peptidoglycan layer by breaking the glycosidic bonds that hold together NAG and NAM …show more content…
As seen in the table above, none of the determined ratios fall within the range. The fact that none of my A260:A280 ratios fell in the range, and only one of my samples fell in the A260 desired range, indicates that there was significant protein contamination in my sample. This could be due to poor pipetting skills, leading to contact with the interface layer when extracting the aqueous layer. In addition, this could also be due to poor mixing, as proper mixing is essential for SDS and sodium perchlorate to denature proteins and inhibit their contact with DNA.
Since only one of my samples had an A260 values that fell within the desired range, the average concentration cannot be calculated. Using the volume of water used to dissolve DNA, and assuming Sample B represents average concentration, the yield of the isolated genomic DNA can be calculated. Yield = average concentration volume of water used Yield = 405g/mL7mL =