Production and Purification of genomic DNA

April,2019 Filipe Fernandes 2470

Lab IVB Denise Barros 2586
Maria Chouzende 2631
Biot23 Beatriz Simões 2634

Abstract
In this assignment we aimed to extract and purify the chromossome of a
Bacillus Cereus strain. First, it was performed a cell lysis, so we can reach to the
nucleoid of the cell, where the genomic DNA is situated, then we did a
solubilization with an organic solvent called phenol, which permits to separate
the DNA based on solubility differences. In order to precipitate the DNA, we
added ethanol to neutralize the DNA’s charges. Finally, it was executed a
agarose gel electrophoresis with a TAE tampon.
In order to determinate the amount of gDNA (genomic DNA) on the Bacillus
Cereus nucleoid, we used absorbance spectroscopy, thereby obtaining 0,1704
mg of wet cells . The yield was obtained in a range of 0,09% DNA/mg wet cells.
Introduction
The primary purpose of genomic DNA purification is its isolation
from the remaining cellular constituents, so that it can be used in
subsequent applications. To do so, DNA purification techniques
involve three major steps:
1.Cellular lysis that allows breaking cell membranes, releasing
all intracellular compounds into the solution After cell disruption, a
cellular homogenate consisting of the compound of interest (in this
case, genomic DNA), contaminating biomolecules and cell
fragments is obtained.
2.DNA solubilization and removal of contaminants through an
organic extraction with a phenol / chloroform / isoamyl alcohol
mixture. The use of phenol (organic solvent) allows to separate the
DNA of the remaining cellular constituents based on differences in
solubility.
3.DNA precipitation this is done by the use of isopropanol
associated with a solution with a high concentration of a cationic
salt. Ethanol induces the structural transition in the acid molecules,
causing them to aggregate, with consequent precipitation.
Precipitation with isopropanol, in addition to concentrating the
DNA, helps remove phenol and chloroform residues from the
sample.
To verify the purity of the extracted genomic DNA and determine its
concentration, we must read the absorbance of the sample at
different wavelengths in a spectrophotometer. To read the
concentration of purified DNA, it is read at a wavelength of 260 nm.

Firstly, cell lysis.

RESULTS
The TE buffer (10 mM Tris-HCl pH 8; 1 mM EDTA) was first used to
maintain the pH of the sample and chelate the divalent metals,
avoiding the action of DNases.
The buffer (20mg / mL proteinase K; 10% SDS) was added to digest a
peptidoglycan cell wall and rupture a cell membrane.
1
2 3
4 5 6 Then, organic extraction (purification).
The use of phenol allows the separation of the DNA from the cellular
constituents based on differences in solubility. The DNA molecules are
negatively charged so they remain in aqueous solution.
An aqueous phase containing the DNA is salts, allowing the DNA to
become less soluble in water and still is ethanol which facilitates the
formation of ionic bonds between the salts and the phosphate groups
present in the DNA molecule, thereby aiding in the precipitation.

Abs White Test 1 Test 2 Average

Abs
CONCLUSION (230nm) 0
Abs
0,201 0,202 0,2015

(260nm) 0 0,425 0,427 0,426

We proceded to read the absorbance of the 260 nm and 280 nm samples, which the reason was Abs
1,945 and 2,109 concluding that the gDNA of out group was contaminanted with RNA. This (280nm) 0 0,218 0,22 0,219
contamination probably happened due the execution of the assignment, as the creation of ar Law of Lambert-Beer:
bubbles during the extration of the supernatant. In addiction, the group calculated the quantity Pure Reasons:
of gDNA using the acquired absorbance , obtaining 21,4 ug um 174 mg of wet cells. We also
calculated the yield using the mass of the initial cells, using the kit of DNA extration , obtaining
the yield of 0.09 ug DNA/MG of wet cells. We can also conclude that the amount of obtained These results show us that the DNA is
gDNA was not in the expected range, because of the mencioned mistakes, and can be also practically pure since it has an appro-
explained by weighting and pipetting mistakes. It should be also noted that on the agarose gel ximate R1 value of 1.8
eletrophoresis it is possible to see that on pit 2, there was less migration because of the weight 0,170mg

and lenght of gDNA , therefore it is less visible.

In conclusion, even though the results were not the expected inicially, the sim of the Yield: In this case it confirms the degree of
contamination, being the value betwe-
assignement was achived , having the possibility of using this knowledge for future assignments.
en the 2 and the 2,2
The methodology used to purify the gDNA in
comparison with other commercial kits (NZY
Microbial gDNA Isolation kit) is the higher con-
centration of pure gDNA by the method used,
however this is much more time consuming than
commercial kits that take about 20 minutes.

REFERENCES
Carla Santos; Laboratório IVB 2018/2019- http://moodle.ips.pt/1819/pluginfile.php/499450/mod_resource/content/3/Guia%20de%20Trabalhos%
20de%20Laborat%C3%B3rio%20IV%20B%2018_19.pdf
Kit de purificação de gDNA: https://www.nzytech.com/products-services/genomic-dna-purification/mb21702/