UNIVERSITI TUNKU ABDUL RAHMAN

Name: Siau Siew Ching

ID No:16ADB05608

Experiment 2: Isolation of Plasmid DNA

Objectives:

1. To isolate plasmid DNA from viable bacterial culture using the alkaline lysis
method.
2. To determine the amount of extracted plasmid from alkaline lysis method by
agarose gel electrophoresis.

Result:

GeneRuler 1kb DNA Ladder

1 2 3 10000kb

3500 kb

Figure 1: Agarose gel electrophoresis of newly extracted DNA plasmid from an

Escherichia coli variety that has the pGLO plasmid ingrained in it. A sample of plasmid
that has been isolated from the same initial bacterial stock is separated into 2 tubes
labelled DNA 1 and DNA 2. After the isolation and distillation of the respective plasmids
the results are determined by electrophoresis with the use of a 1kb DNA Ladder for
scale.The number above each lanes 1: represents the DNA ladder of the plasmid DNA,
lanes 2: samples DNA 1,lanes 3: samples DNA 2

Answer all the following questions:

1. Explain the functions of Solution I, II and III, phenol:chloroform, and

absolute ethanol in plasmid extraction.

Solution I contains glucose, Tris, and EDTA. Glucose is added to maintain the osmotic
pressure so that the cell would not lyse. Tris is a buffering agent used to maintain a
constant pH ( = 8.0). EDTA protects the DNA from degradative enzymes ( DNAses)
damaging the plasmid and also helps by destabilizing the cell wall(Oswald, 2007)[3].

Solution II is the cell lysis Solution, which contains sodium dodecyl sulfate (SDS) and
sodium hydroxide, is then added. SDS is a detergent that dissolves phospholipid
membranes leading to the lysis of the bacterial cell wall. The sodium hydroxide raises the
pH, which not only denatures cell proteins, but also denatures DNA to single strands and
degrades high molecular weight RNA(Oswald, 2007)[3]..

Solution III act as a neutralization solution, containing potassium acetate and acetic acid.
Acetic acid use to neutralize the pH so the DNA renatures while the SDS precipitates
along with cellular debris. The chromosomal DNA also precipitates because it is attached
at several sites to cell membrane proteins. A spin in the centrifuge separates most of the
precipitated molecules from the plasmid DNA still in solution(Oswald, 2007)[3].

A biphasic emulsion forms when phenol chloroform are added. The hydrophobic layer of
the emulsion will then be settled on the bottom and the hydrophilic layer on top by
centrifugation (Tan and Yiap, 2017). Phenol:chloroform act as organic solvent to remove
proteins, lipids, carbohydrates, and cell debris through extraction of the aqueous phase
with the organic mixture of phenol and chloroform (Tan and Yiap, 2017). Water-soluble
DNA has less density then phenol and chloroform thus will separates into the aqueous
phase. At the same time, the proteins will be denatured by the organic solvents and stayed
in the organic phase which is denser. Chloroform was added in to increase the density of
the organic phase further prevent phase inversion and reducing the interphase, which was
the border between the two phases The interphase consists of partially denatured proteins
or DNA and partially denatured DNA binding proteins that were still clinging to the
DNA(Plank, 2007). When the interphase was accidentally pipetted together with the
aqueous phase, it will decrease the purity of the sample. But if too little aqueous phase
was pipetted, the yield of plasmid DNA will decrease (Plank, 2007).

Absolute ethanol was added to desalt and concentrating DNA in aqueous solution.
Ethanol alter the structure of the DNA to allow the DNA molecules were aggregated
and precipitated by reduce the hydrophilic property of(Oswald, 2007)[1]. . Ethanol
dissolved most salts and small organic. Ethanol also cause the nucleic acid to aggregate
by decrease the hydrophilic properties(Oswald, 2007)[1]..

2. Comment on the yield and purity of your isolated plasmid DNA.

Graph 2.1 The samples DNA1 was measured on Nanodrop with using TAE
buffer as blank.
 Graph 2.2 The samples DNA 2 was measured on Nanodrop with using TAE
buffer as blank.

From the nanodrop spectrophotometer , the graphs showed both samples the yields of
DNA plasmid with amounts for samples DNA 1 and DNA 2 are 965.8ng/l and
175.2ng/l respectively. DNA plasmid derived from the cell samples consider as high
yield level.

The ratio of absorbance at 260 nm and 280 nm is used to assess the purity of DNA and
RNA. The absorbance ratio 260/280 in nanodrop spectrophotometer detect the level of
protein contamination in the samples. Protein has a light absorbance at 280nm. 260:280
ratio has high sensitivity for nucleic acid contamination in protein. Thus, lower A260/280
values may indicate protein contamination(Ogt.com, 2017). A ratio of ~1.8 is generally
accepted aspure for DNA. 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.
Whereas 260:230 ratio has low sensitivity for protein contamination due to protein show
low light absorbance at 260nm and 280nm. Thus,lower A260/230 values indicate
contamination with salts or some solvents(Ogt.com, 2017).

From the results DNA 1 shows 260/280 at 2.12, and 260/230 at 2.36. whereas for DNA 2
showing 260/280 at 2.14, and 260/230 at 2.36. Both show nearly identical results and
have a relatively high purity of DNA plasmid.

3. How many DNA fragments were observed from the lanes loaded with the
extracted plasmid DNA? Explain what those bands are and how they differ
from each other.

From the agarose gel image, two DNA fragments which is supercoiled plasmid DNA and
the relaxed plasmid DNA were observed from the lanes loaded with the extracted
plasmid. The supercoiled plasmid DNA migrate fast than the relaxed plasmid DNA due
to the size of supercoiled plasmid DNA is much more smaller. Moreover, supercoiled
DNA is more compact and the smaller molecular size can pass through the agarose gel
easily than the relaxed molecule which is less coiled and has larger size.

4. From your gel image, state whether it is possible for you to accurately
estimate the size (in base pairs) of the extracted plasmid DNA by referring to
the DNA ladder. Justify your answer.

From the gel image, it is possible to estimate approximately the size of the extracted
DNA by referring according to the DNA ladder. This is because the bands is linear with
the level of DNA ladder at 10000kb and 3500kb.The supercoil plasmid migrate far than
relaxed DNA and level at 3500kb whereas the relaxed plasmid level at 10000kb. This is
because supercoil plasmid is compact and thus has smaller size of base pair than the
relaxed plasmid DNA.
References

Barbas, C., Burton, D., Scott, J. and Silverman, G. (2017). Quantitation of

DNA and RNA. [online] Cold Spring Harbor Protocols. Available at:
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