Introduction

In Olden Days, it
was common assume that proteins would carry the genetic material, because DNA
was just way too dull to show any hereditary correlation. On the other hand,
proteins had higher complexity and variety, thus it had potential to show
hereditary correlation. The discovery of DNA wasn’t made until 1953, when Watson
and Crick published the double helix structure of DNA, and further studies established
the importance of DNA as a hereditary structure.  After multiple decades of research DNA can be
thought as the structure for all life on Earth, because DNA carries genetic
intelligence which is responsible for function, development,
reproduction(replication) of all known living organisms.1 Due to
highly integrated function of DNA this led to tremendous growth and
advancements in the field of DNA and its application.

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One of the biggest
problem with isolation of DNA in vivo
is that there is a high concentration of other components in the cell.
Scientist have developed methods to isolate genomic DNA to determine the
chemical structure of DNA by avoiding the contaminants of the cell. These
extraction methods assist scientists to produce the individual human genome,
detecting paternity, genetic causes of disease, and eventually development of
the genetically engineered organisms to create products such as antibiotics and
hormones.2

In this
experiment, DNA was extracted and isolated to experimentally determine the size
of the E. coli genome. In order to
isolate genomic DNA, HB101 E. coli
were harvested, lysed, and the DNA was purified from the cell extract by using
the phase extraction.  The amount of DNA
isolated was determined using the Genesys 5 Spectrophotometer, and the cell density
was calculated from the HB101 strain on the incubated LB plates assuming the
total culture volume of 75 mL. The experimental value of the collected DNA was
then compared to the theoretical values of the HB101 strain of E. coli.

Methods

A single colony of HB101 was
inoculated in the BHI rich medium, and then the overnight growth of the
colonies into the 75mL of BHI rich medium. Serial dilution was performed to
count the number of cell growth on the LB plates. After plating the cells with
different dilution factors on LB plates, the plates were incubated at 37°C for growth. The number
of cells were counted on the LB plates, while the initial cell culture volume
of 75mL to determine the cell density. Two Chloroform-resistant centrifuge
tubes were prepared from rest of the remaining E.coli culture. After centrifuging, 2 tubes were filled with HB101
cells, the pellet was then suspended into saline-EDTA, lysozyme, and RNAase, and then incubated the cells in water bath.
After incubation added SDS, sodium perchlorate, and chloroform-isoamyl alcohol.
After mixing carefully, centrifuged the tubes to obtain the separation of
phases for further extraction of DNA. After extracting the aqueous layer from
the top of the centrifuged tubes, added ethanol and obtained the gelatinous
“glob” using the glass rod. Then suspended the extracted DNA into 4 mL dH20.
Prepared 4 different cuvettes with different dilution factors, and then used
the Genesys spec to determine the absorption at 260nm and 280nm for the
samples.

Results

The absorbance of the extracted DNA
was measured at both 260nm and 280nm. Then the ratio was used to determine the
purification of the sample. The Precipitated DNA was collected on the glass
rod, and then the spooled DNA was suspended into 4 ml dH20.

 

Table #1: Absorbance values at 260nm and 280nm for genomic
DNA from purified E. coli, and the
diluted and final concentration of ug of DNA/mL, along with the experimental ug
of DNA in 4 mL of dH20.

Sample

Dilution factor

A260

A280

A260:A280

Concentration of Dilution (ug/mL)

Final Concentration (ug/mL)

Total DNA (ug)

A

10

0.847

0.395

2.14

42.4

424

1696

B

50

0.179

0.088

2.03

8.95

448

1792

C

100

0.100

0.051

1.96

5.00

500

2000

Average

 

 

 

2.04

 

457

1829

Due to high ratio for uracil, a
260/280 ratio above 2 is often suggestive of RNA contamination and then the
total DNA was 1829 ug was compared to the theoretical DNA.

 

 

 

 

Table #2: Dilution factor for the
cuvettes made for the Genesys spectrophometer to measure the Absorbance levels
at A260 and A280.

Sample

dH20 (uL)

DNA Sample (uL)

Final Volume (uL)

Dilution Factor

Blank

1000

0

1000

N/A

A

900

100

1000

10

B

980

20

1000

50

C

990

10

1000

100

 

Table #3: Cell Density after assuming the initial culture of
75 mL, at different diluted plates.

LB plate

# of colonies

Volume plates (mL)

Dilution Factor

Cell Density (cell/mL)

105

Lawn

0.1

10

N/A

106

90

0.1

10

9.0*108

 

Total number of cells which the DNA
was isolated from was 6.75*1010 cells. Both plates should give
similar results, because the only variation was the dilution factor. If cell
density of 105 diluted LB plate was used then the initial number of
cells should be the same as the 106 diluted LB plate.

Assuming the 100% effectiveness of
the DNA isolation the experimental value is 2.70*10-14 grams/cell.

 

Discussion

In order to
compare the experimental and the theoretical value of mass of DNA per cell, the
assumption was made that the E. coli
chromosome contains 4.6*106 nucleotides with an average molecular
weight of 650 g/mol. The sample calculation is shown in appendix. The
theoretical value for the DNA mass per cell was 4.32*10-15 g/cell. The
theoretical value differed from the experimental value by a factor of 6. DNA
samples was contaminated by the RNA, A260:A280 was higher than 2.0, which would
overestimate the mass of DNA isolated, since the experimental DNA was higher
than the theoretical DNA. Another factor that affected the cell number was the
growth of the colonies from the exponential phase into the death phase. Only
the live cells had the potential to grow on the LB plates. Therefore, the cell
density would be underestimated and that would underestimate the total number
of cells and that would create an overestimate of the mass of DNA per cell.
Both the RNA contamination and growth factor could account for the higher
experimental value.

            Overall
the DNA extraction is fairly reasonable technique because it was almost in
compliance with the theoretical values. All the reagents worked in compliance
to have an actual DNA extraction. For instance, EDTA served as the chelating
agent, lysozyme degraded the cell b-(1,4)-glycosidic linkages, RNase degrades
RNA, Sodium perchlorate which inhibits the interaction between the proteins and
DNA, SDS aids in lysis and denature proteins, ethyl alcohol helped in spooling
the DNA samples. Due to a contamination of RNA, there could be a possibility of
not adding enough RNase, but the contamination was not that high, it was almost
pure DNA. The dilution factors were perfect, as the graph of absorption and
concentration showed a perfect linear correlation.

            For
further studies, we could have potentially used the isolated DNA to run the PCR
and gel electrophoresis to obtain the better results.

 

Appendix

 

Sample Calculation
for Cell Density:

 

Total Number of
cells:

 

Assuming 100%
effective of the DNA isolation:

 

Sample Calculation
for the Dilution Factor

 

General Formula: –

Sample Calculation for cuvette A:

 

 

 

Sample Calculation
for the Ratio of A260:A280 and concentration of the Dilution and Final
Concentration

 

Ratio for Sample A:

 

Concentration
of Dilution: –

 

A= 0.847

e=0.020

L= 1 cm

 

Concentration =42.35

 

Final Concentration
= Diluted Concentration * Dilution Factor

                                    42.35*10=423.5

 

 

References

 

 

1)    BMB
442 Laboratory Manual

 

2)    “What
Is DNA? – Genetics Home Reference.” U.S. National Library of Medicine,
National Institutes of Health. Science (2018).