Derek Wong
Biology 275L - 03
Lab 8
Isolation of genomic DNA from Drospophila Melanogaster
and
plasmid DNA from E. Coli.
Introduction
Deoxyribonucleic Acid (DNA) consists of a nucleotide polymer used to create a cells genetic makeup. This genetic makeup determines what type of characteristics a particular cell (and organism) will have.
Plasmids are small fragments of extrachromosomal DNA that carry pieces of genetic information which can be have genetic information inserted, replicated, and studied in them.
There are five steps in isolating DNA. The first is to lyse the cell by use of a chemical lysis or a homogenizer. This will break the cell’s membrane as well as the nuclear membrane releasing the DNA. The second step is to remove the RNA and proteins by addition of an enzyme that hydrolyzes RNA (RNAses). The third is to separate the DNA from the aqueous solution with alcohol and to wash the DNA precipitate in 70% ethanol. The fourth step is to hydrate the DNA with water and EDTA. The final step is to quantify the amount of DNA by use of a spectrophotometer reading at 260nm and at 280nm. Besides using the spectrophotometer, one can also use electrophoresis and determine the quality and quantity from that. The darker the bands, the more DNA there is, and the less bands and smearing there are the higher the quality of DNA was obtained.
In this experiment, the genomic DNA of the Drosophilia Melanogaster and the plasmid DNA from E. Coli will be isolated and then the quality and quantity will be determined using spectrophotometry and electrophoresis.
Results:
Table 1: Spectrophotometer results of the genomic DNA and the plasmid DNA.
|
|
260nm Absorption |
280nm Absorption |
Ratio |
|
Genomic DNA |
0.769 |
0.552 |
1.3939 |
|
Plasmid DNA |
0.237 |
0.176 |
1.3479 |
Table 1 show the spectrophotometrically the quantity and quality of the genomic DNA from Drosophilia Melanogaster, and the plasmid DNA isolated from E. Coli.
Table 2: The concentration estimation of genomic DNA and plasmid DNA.
|
|
260 nm Absorption |
Concentration (Estimated) |
|
Genomic DNA |
0.237 |
296.25 ug/mL |
|
Plasmid DNA |
0.769 |
961.25 ug/mL |
Table 2 shows the concentration estimate by inserting the 260nm absorption reading into the given equation of: 260nm Abs X 50 (ug/ml)/(OD) X 25 = DNA concentration.
Figure 1 (see next page for actual copy): Agarose gel eletrophoresis of isolated genomic DNA from Drosophilia Melanogaster and plasmid DNA isolated from E. Coli. This figure shows figuratively the quality and quantity of the DNA isolated from both groups as well as the DNA marker (to the left). [bp = base pairs]
Lane 1: Our Plasmid DNA sample, Lane 2: Our Genomic DNA sample
Lane 3: Standard Marker
Lane 4: Other groups E. Coli sample, Lane 5: Other group Genomic DNA sample
Discussion:
In this lab, genomic DNA was isolated from Drosophilia Melanogaster and plasmid DNA was isolated from E. Coli and the results were analyzed via spectrophotometer and agarose gel electrophoresis.
The results for the spectrophotometer are shown in both Table 1 and Table 2. Table 1 shows the raw data obtained from the spectrophotometer at 260nm and at 280nm absorption. The ratio is taken from the reading at 260nm divided by 280nm. The closer the ratio is to two, the purer the sample is. If the sample’s ratio is greater than 2, then there is RNA contamination, meaning that in the DNA isolation technique, not all of the RNA was separated out. If the ration I less than 1.6, as in this case, then protein contamination is indicated meaning that purification was not complete and there was still excess solution when the gels were ran. Table 2 shows the estimated DNA concentration for the genomic and plasmid DNA. In the E. Coli, it was estimated that there was 296.25 ug/ml compared to the given of ~225 ug/ml. In the genomic DNA, it was estimated to be 961.25 ug/ml compared to the given of ~ 787 ug/ml. Since results from both DNA sample is larger than the given, it can also be concluded that there is protein contamination and/or some other type of contamination that caused there to be a reading of more DNA then there actually is.
The agarose gel electrophoresis manifests the quantity and quality of the DNA samples by the intensity and number of bands. By comparing the bands intensity (how bright it is) the quantity can be relatively compared. The plasmid DNA was observed to have more intense bands and it can be concluded that there was more plasmid DNA than genomic DNA in the samples ran. In the sample ran on farthest two left lanes (our samples), there was smearing indicating that the samples were impure (which corresponds to our results). The other group was observed to have more definitive lines indicating a more pure sample. When comparing the genomic DNA and the plasmid DNA, the intensity and number of bands differs. Since genomic DNA is a larger molecule it has only one band (above 10,000 bps). The E.Coli samples showed three bands indicating that plasmid DNA has three components (at above 10,000 bps, 4,000 bps, and at 2,000 bps). The main difference between genomic DNA and plasmid DNA is that plasmid DNA are extra-chromosomal, smaller and circular versus genomic DNA which is much larger. These differences in genomic and plasmid DNA will cause a difference in the way plasmid DNA is isolated from cells in that during the lysis stage, lysis is done by sodium hydroxide for isolation of plasmid DNA which denatures both plasmid and chromosomal DNA instead of a homogenizer. This denaturation will cause DNA to lose its supercoiled structure.
In this experiment, ethidium bromide was used as a
fluorescent dye in the gel eletrophoresis
visualization of DNA by binding to DNA.
However, it is a potent mutagen because of its ability to intercalate
with DNA, allowing it to interfere with the transcription and translation of
DNA thus allowing mutagenesis to occur. To protect oneself, one should wear a
lab coat, eye protection, and nitrile gloves when
working with ethidium bromide.