TITLE: EXTRACTION OF GENOMIC AND PLASMIDS DNA FROM BACTERIAL

CELLS
NAME: SEIMA BB
STUDENT NR: 31490778
TOTAL: 75
RUBRIC
Mark

1 Opsomming/Abstract [10]

2 Relevansie van inleiding / Relevance of introduction [10]

3 In-teks verwysings / In-text referencing [5]

4 Leisinne vir figure en tabelle / Leading sentences for

figures and tables [5]

5 Tabel en figuur beskrywings (Volledigheid) / Table and

figure descriptions (Completeness) [10]

6 Interpretasie van resultate / Interpretation of results [10]

7 Relevansie van bespreking / Relevance of discussion [10]

8 Gevolgtrekking / Conclusion [10]

8 Bronne / Reference list [5]

Totaal/Total [75]
ABSTRACT/SUMMARY
This experiment involved the isolation of E. Coli genomic DNA followed by an
assessment of its purification. The isolation process involved cell lysis followed by
the removal of RNA, proteins, and lipids. Once separated from these components,
the DNA was precipitated by ethanol and purity and concentration were determined
using Genesys spectrometry. This device determines the absorbance of the
substance being tested at different wavelengths. Concentration is calculated by
observing the absorbance at 260 nm (A260) which lets us know how much double-
stranded DNA was present in the original sample. This A260reading can also be
used along with an A280reading to assess the purity of the DNA. Less pure DNA is
contaminated with either RNA or proteins which occurs due to errors during the
isolation process. The absorbance readings are used to create a ratio (A260: A280).
Due to RNA and protein absorbing different amounts of light than DNA at these
wavelengths, a ratio out of the range for pure DNA indicates contamination. The
process carried out in this experiment was done in the most manual setting as
possible. As technology advances, more automated ways of isolating DNA as well as
other cellular components have been developed. Creating more automated methods
has resulted in more time saved as well as the generation of more reliable results,
because the manual method consists of many more steps and reagents, there is
greater room for error. Automated methods may continue to develop to become
100% automated and other improvements like portability could be incorporated.

INTRODUCTION
DNA once the most difficult cell to analyse, but today it has become one of the
easiest. With technology and knowledge it is now possible to produce an unlimited
number of copies and determine its nucleotides. By isolating and purifying DNA it
becomes possible to determine the biochemical structure and its function in an
organism. (Chaudhuri S. 2005)
Bacterial cell genomes consist of a single chromosome (sometimes more than one)
and some extra-chromosomal DNA molecules called plasmids. The process of
extracting DNA from bacterial cells can be done using an organic method or
commercial DNA extraction kits based on spin column technology. The aim of this
experiment is to extract and isolate DNA from bacterial sample, purify and quantify
the DNA extract. When extracting the DNA, an organic method or commercial DNA
extraction kit (Quick-DNA Mini-prep Isolation Kit) based on spin column technology
and centrifugation, the separation was achieved through Agarose Gel-
Electrophoresis. In order to validate the purification process, spectrophotometry was
performed at different wavelengths and absorbance values were recorded.

This experiment consists of E.Coli cells being cultured and lysed to isolate and purify
their genomic DNA. The proteins, RNA and lipids were removed through phase
extraction. The DNA was obtained by spooling and dissolving into ethanol which was
then diluted and quantified using Genesys spectrometry so that we can know how
pure our DNA is and also obtain the absorbance readings and the total DNA
concentration present. Cell density was also calculated using colony counts on LB
plates that were made using E.Coli and saline dilution

MATERIALS AND METHODS

A sample Up to 1 ml of bacterial culture was prepared and can be used for the
preparation depending on density of culture, culture medium, and bacterial strain.
The sample was centrifuged up to 1 ml culture for 5 min at 8,000 x g. The
supernatant was poured into the supplied beaker. Pre-lyse sample Re-suspended
the pellet in 180μl Buffer T1 by pipetting up and down, 25 μl Proteinase K was
added. The mixture was vortexed vigorously and incubated at 56°C for 1 hour, the
sample was lysed and put in a vortex. 200μl Buffer B3 was added, vortexed
vigorously and incubated at 70 °C for 10 min. DNA binding conditions were adjusted
and 210μl ethanol was added (96 – 100 %) to the sample and vortex vigorously.
After addition of ethanol a stringy precipitate may become visible. Bind DNA for
each sample, one NucleoSpin tissue column was placed into a Collection Tube. The
sample was applied to the column and centrifuged for 1 min at 11,000 x g. The flow
through was discarded and placed the column back into the collection tube. Silica
membrane was washed and 50μl Buffer BW was added. Centrifuged for 1 min at
11,000 x g. Discard flow-through and place the column back into the Collection Tube
10. 600μl Buffer B5 was added to the column and centrifuged for 1 min at 11,000 x
g. Discard flow through and place the column back into the Collection Tube. Dry
silica membrane Centrifuged the column for 1 min at 11,000 x g. Residual ethanol
was removed during this step. Elude highly pure DNA Place the NucleoSpin tissue
column into a 1.5 ml micro centrifuge tube and 50μl pre-warmed Buffer BE (70°C)
was added and Incubated at room temperature for 1 min. Centrifuge 1 min at 11,000
x g to collect the DNA sample in the clean tube. 13. The DNA for agarose gel
electrophoresis was kept and concentration determination.

Agarose gel electrophoresis of the DNA

Prepare a 1 % agarose gel was prepared, 5 µl of the DNA and 2 µl of loading buffer
was mixed together, containing Gel-Red as dye and transfer to one of the wells in
the gel. In addition, loading dye was added and load an appropriate size marker (1kb
DNA marker, Fermentas). Electrophoresis conditions: 45 minutes at 80 V. 5. View
with gel documentation equipment and photograph the gel.

UV Characterisation of the DNA

A 260 nm and 280 nm and 230 nm OD reading of the DNA using a Nanodrop
spectrophotometer was obtained, 2 µl distilled water was used as blank. The data
was used to calculate the concentration of the DNA 4. The quality of the DNA
preparation was determined by calculating the A260/A280 ratio and the A260/A230
ratio.
RESULTS
DNA was isolated and purified from bacterial cells using a commercial DNA isolation
kit. After performing spectrophotometry of the concentrated DNA, the following
results were obtained:

Figure 1: Agarose Gel with genomic DNA

As the gel runs, shorter pieces of DNA travelled through the pores of the gel matrix
faster as compared to the longer ones.

Figure 2: Agarose Gel with plasmid extracted from E.coli.

As the gel runs, shorter pieces of plasmids travelled through the pores of the gel
matrix faster as compared to the longer ones.
 Table 1: Concentrations of DNA with their respective absorbance
Sample ID ng/ul A260 A280 A230 260/280 260/230
Gel 1
1 121.55 2.431 1.201 1.198 2.024 2.029
2 158.2 3.164 1.531 1.445 2.066 2.189
3 200.35 4.007 1.915 1.773 2.029 2.260
4 336.05 6.721 3.345 3.185 2.009 2.110
5 102 2.04 1.034 1.360 1.972 1.500
6 36.6 0.732 0.396 0.370 1.848 1.978
7 166 3.32 1.64 1.523 2.024 2.179
8 44.8 0.896 0.474 0.417 1.890 2.148
9 216.95 4.339 2.057 1.895 2.112 2.289
10 36.55 0.731 0.429 0.717 1.703 1.019
11 80.3 1.606 0.837 0.945 1.918 1.699
12 132.45 2.649 1.308 1.299 2.025 2.039
13 49.65 0.993 0.531 0.725 1.870 1.369
14 187.8 3.756 1.798 1.755 2.088 2.140
15 266 5.23 3.125 2.531 1.673 2.066
Gel 2
1 104.4 2.088 1.067 1.123 1.956 1.859
2 194.1 3.882 1.924 1.765 2.017 2.210
3 161.6 3.232 1.573 1.449 2.054 2.23
4 45.3 0.906 0.478 0.477 1.895 1.899
5 88.7 1.774 0.887 0.939 2 1.88
6 148.05 2.961 1.542 1.526 1.920 1.940
7 76.45 1.529 0.808 0.916 1.892 0.808
8 154.7 3.094 1.483 1.400 2.086 2.21
9 81.65 1.633 0.869 0.939 1.879 1.739
10 106.5 2.13 1.107 1.170 1.924 1.820
11 188.95 3.779 1.785 1.643 2.117 2.300
12 159.9 3.198 1.573 1.545 2.033 2.069
13 79.75 1.595 0.804 0.794 1.983 2.000
14 81.7 1.634 0.807 0.729 2.024 2.241

DISCUSSION
The process of Quantification is a very crucial process, because with it one is able
tell whether a sample is a pure sample be it of DNA or RNA (Bakken, 1995). And
therefore in order two ratios must computed, the ratio of A260/A280 and of
A260/A230. A260/A280, the ratio of absorbance at 260 nm and 280 nm is used to
assess the purity of DNA and RNA. A ratio of ~1.8 is generally accepted as “pure” for
DNA; a ratio of ~2.0 is generally accepted as “pure” for RNA. If the ratio is
appreciably lower in either case, it may indicate the presence of protein, phenol or
other contaminants that absorb strongly at or near 280 nm. A260/230, this ratio is
used as a secondary measure of nucleic acid purity. The 260/230 values for “pure”
nucleic acid are often higher than the respective 260/280 values. Expected 260/230
values are commonly in the range of 2.0-2.2(William et.al, 1997). If the ratio is
appreciably lower than expected, it may indicate the presence of contaminants which
absorb at 230nm. From the results and computation, the sample clearly has
contaminants and this is due to the error that might have occurred in washing step
during the isolation of DNA.

CONCLUSION
The aim of the experiment is to isolate DNA from a bacterial sample, purify the DNA
extract and quantify the DNA extract, is successfully accomplished, Despite the
errors that might have affected the results, almost all the students got the same
results thus the experiment is reliable and can be repeated to obtain results. .
Therefore Quick-DNA Miniprep Isolation Kit is the easiest DNA isolation kit for rapid
isolation of total DNA.
REFERENCING
1. Bakken, L.R. 1995, Separation and purification of bacteria. Applied Environmental
Microbiology. 49, 143-169

2. Brisson-Noel, A. et al., Diagnosis of tuberculosis by DNA amplification in clinical

practice evaluation. Lancet, 1991, 338, 364– 366.

4. Ray Chaudhuri, S., Kundu, S. and Thakur, A. R., Microbial biodiversity screening
in East Calcutta Wetlands. In Proceedings of International Seminar on Frontiers of
Basic and Applied Molecular Biology, India, 2005, pp. 64–71

3. William W. W, Mackey K, Chomczynski P., 1997, Effect of pH and Ionic Strength

on the Spectrophotometric Assessment of Nucleic Acid Purity: BioTechniques
22:474- 481.