UEMB 4173 MOLECULAR BIOLOGY
REPORT 1
Extraction of E. coli Chromosomal and Plasmid DNA, Ultraviolet
Measurement of DNA and DNA Characterization by Agarose Gel
Electrophoresis
NAME: Soh Kk Ling
STUDENT ID: 1405497
COURSE: BI
Abstract
The objectives for this experiment are to study the extraction of chromosomal and
plasmid DNA, to determine the size of extracted E.Coli chromosomal and plasmid
DNA using gel electrophoresis, to study the relationship between DNA absorbance and
wavelength, and to study the hyperchromic effect of DNA using UV transilluminator.
Size of chromosomal DNA was compared to standard lambda DNA, and both their
sizes were determined by plotting and extrapolating a semi-log graph of DNA size
against distance travelled across agarose gel, by referring to 1kb DNA ladder, the sizes
of the DNAs can be determined. The size of extracted chromosomal DNA is 14kb.
A260 and A280 of double stranded DNA were measured to show that absorbance at
260nm is higher than 280nm; the A260/A280 ratio is then used to determine the purity
of extracted chromosomal DNA. The A260/A280 ratio in the experiment is 1.6761,
which is near the expected value, 1.8. A260 of DNA at different extent of denaturation
was measured, the relationship of increasing absorbance with increasing denaturation
has proven the hyperchromic effect. Principles of DNA extraction, hyperchromic effect
and determination of DNA purity were discussed in this report. The factors that may
affect A260/A280 ratios were also discussed.
Introduction
The ability to extract DNA is of primary importance to studying the genetic causes of
disease and for the development of diagnostics and drugs. It is also essential for carrying
out forensic science, sequencing genomes, detecting bacteria and viruses in the
environment and for determining paternity. Deoxyribonucleic acid (DNA)
extraction involves separating the nucleic acids in a cell away from proteins and other
cellular materials.
The extraction of DNA generally follows three basic steps: 1. Lyse (break open)
the cells. 2. Separate the DNA from the other cell components. 3. Isolate the DNA. The
cell membrane is disrupted by any of the following methods: using heat to increase
fluidity, dithiothreitol (DTT) to reduce disulfide bonds, or a detergent, such as sodium
dodecyl sulfate (SDS), to disrupt the membrane. Proteins, including nucleases, are
inactivated by heat denaturation or by digestive enzymes to cut them up. The
temperature must be kept below 60°C and the period must be kept sufficiently short
(15 to 20 minutes) if nondegraded, high-molecular-weight DNA is required. If the DNA
remains in the aqueous phase, it is separated from the other cellular materials including
proteins and lipids by centrifuging the latter to the bottom of the tube or partitioning
them in organic solvents (Elkins, 2012).
Originally evolved from bacteria, plasmids are extrachromosomal genetic
elements present in most species of Archae, Eukarya and Eubacteria that can replicate
independently. Plasmids are circular double stranded DNA molecule that are distinct
from the cell chromosomal DNA. Plasmids present in the bacterium differ in their
physical properties such as in size (kbp), geometry and copy number. The structure and
function of a bacterial cell is directed by the genetic material contained within the
chromosomal DNA. In some cases plasmids are generally not essential for the survival
of the host bacterium. Although not essential, plasmids contribute significantly to
bacterial genetic diversity and plasticity by encoding functions that might not be
specified by the bacterial chromosomal DNA. Antibiotic resistance genes are often
encoded by the plasmid, which allows the bacteria to persist in an antibiotic containing
environment, thereby providing the bacterium with a competitive advantage over
antibiotic-sensitive species. As a tool, plasmids can be modified to express the protein
of interest (e.g., production of human insulin using recombinant DNA technology).
Plasmids have served as invaluable model systems for the study of processes
such as DNA replication, segregation, conjugation, and evolution. Plasmids have been
pivotal to modern recombinant DNA technology as a tool in gene-cloning and as a
vehicle for gene-expression.
Materials and Methods
1.1 Chromosomal DNA Extraction and characterization by Agarose Gel
Electrophoresis
1ml of an overnight E.coli culture was added to a 1.5ml micro centrifuge tube and was
centrifuged at 12000 rpm for 2 min. The supernatant was removed. The cell pellet was
suspended in 600 μl of Lysis Solution (LS) and then incubated at 80°C for 5 min to lyse
the cells. The tube contents were cooled to room temperature. 600 μl of phenolchloroform-isoamyl alchohol (PCI) was added to the cell lysate, mixed using vortex
device for 20 seconds and then centrifuged at 12000 rpm for 3 min. The supernatant
containing the DNA was transferred to a clean 1.5ml microfuge tube containing 600 μl
of room temperature absolute ethanol and 300 μl potassium acetate, the contents were
gently mixed by inversion and then centrifuged at 12000 rpm for 2 min. The supernatant
was poured off gently and the tube was drained on clean absorbent paper. 600μl of room
temperature 70% ethanol was added to the supernatant and gently inverted for several
times to wash the DNA pellet. The mixture was centrifuged at 12000 rpm for 2 min.
The ethanol was poured off gently and drained on clean absorbent paper. The pellet was
air-dried for 10–15 min. 100 μl of TE buffer was added to the tube and the DNA was
rehydrated by incubating at 65°C for 1 hr.
0.7% of agarose solution was prepared by adding agarose powder to 1X TBE
buffer. The solution was boiled using microwave oven. Nucleic acid stain was then
added. The solution was let to cool and then poured into the gel tray. A comb was
inserted and the gel was let to solidify. The gel was placed in a gel tank and 1X TBE
buffer was poured in. 2 drops of 2 μl of 6X loading dye were pipetted on top of a
parafilm. 15 μl of each DNA was mixed with 2 μl of 6X loading dye. 3 μl of 1 kb DNA
ladder was pipetted into the first well. 15 μl of standard lambda DNA was pipetted into
the second well. Two 17 μl DNA sample mixtures were pipetted into third and fourth
wells of the agarose gel. The electrophoresis was ran at 80 V for 30 min. The gel was
removed from the gel tank and the result was viewed using a UV transilluminator.
1.2 Ultraviolet Measurement and Denaturation of Isolated DNA
Each of the extracted DNA and standard λ DNA was diluted with 0.9 ml of the TE
buffer. 200 μl of the solution was transferred to a quartz cuvette and A260 was
determined. (Reading was blanked first with TE buffer before measuring DNA
solution). If the A260 is greater than 1, the sample was quantitatively diluted until the
absorbance reading is between 0.5 and 1.0. The A260 and A280 on the same DNA
sample were determined and recorded. 2 ml of each DNA solution in TE buffer was
prepared at a DNA concentration of 20 μg/ml. The A260 was measured and recorded.
0.5 ml of the DNA solution was transferred into each of three test tubes. One tube was
maintained at room temperature and the other two were placed in a 90°C water bath for
15 min. After the incubation, the tubes were removed. One heated tube was quick-
cooled in an ice bath and the other heated tube was cooled slowly to room temperature
over a period of about 1 hr. Final A260 readings on each of the three tubes were
measured and recorded. The A260 /A280 ratio was calculated. The A260(T)
/A260(25°C) ratio for each of the three tubes was calculated.
1.3 Isolation of Plasmid DNA and characterization by Agarose Gel Electrophoresis
1ml of the E.coli culture was transferred to a micro centrifuge tube and was centrifuged
at 12000 rpm for 1 minute. The supernatant was discarded. 100 μl of ice cold alkaline
solution I and 1 μl of RNAse were added into the pellet. The contents in tube were
mixed by vortex for 3 min. 200 μl of alkaline lysis solution II was added. The tube was
inverted for a few times and placed in ice for 5 min. 150 μl of ice cold alkaline solution
III was then added. The tube was again inverted for few times and placed in ice for 5
min. The tube was centrifuged at 12000rpm for 10 min and the supernatant was
transferred to a new micro centrifuge tube and then left in ice. 500 μl buffered phenolchloroform was added to tube. The tube was vortexed for 1 min and centrifuged at
12000 rpm for 10 min. Supernatant was transferred to a new tube. 1 ml of absolute
ethanol was added to the supernatant in tube. The tube was vortexed and left standing
for 2 min at 25°C. The tube was centrifuged at 12000 rpm for 10 min and the
supernatant was discarded. 1 ml of 70% ethanol was added to the tube and centrifuged
at 12000 rpm for 5 min. The supernatant was discarded and the pellet was dried. 30 μl
TE buffer was added to the pellet.
0.7% of agarose solution was prepared by adding agarose powder to 1X TBE
buffer. The solution was boiled using microwave oven. Nucleic acid stain was then
added. The solution was let to cool and then poured into the gel tray. A comb was
inserted and the gel was let to solidify. The gel was placed in a gel tank and 1X TBE
buffer was poured in. Two pBR322 plasmid were pipetted into the first and second
wells. One drop of 2 μl of 6X loading dye were pipetted on top of a parafilm. 15μl
plasmid DNA were mixed with 2 μl of 6X loading dye. The mixture was pipetted into
the third well of the agarose gel. The electrophoresis was ran at 80 V for 30 min. The
gel was removed from the gel tank and viewed using a UV transilluminator.
Results
1.1 DNA Extraction and characterization by Agarose Gel Electrophoresis
Figure
1:
Characterisation
of
E.CoLi
chromosomal DNA. Lane 1: BioLabs 1kb DNA
ladder. Lane 2: Standard Lambda DNA. Lane 3:
E.CoLi chromosomal DNA sample 1. Lane 4:
E.CoLi chromosomal DNA sample 2, with some
smear at the positive end.
By referring to Appendix A, the semi-log graph of DNA size against distance travelled
across agarose gel, the size of standard lambda DNA is 20kb, and chromosomal DNA
sample is 16kb.
1.2 Ultraviolet Measurement and Denaturation of Isolated DNA
A260 of DNA sample control: 0.2737
A280 of DNA sample control: 0.1633
A260/A280 ratio of DNA sample control: 1.6761
A260 of DNA sample without cooling: 0.7808
A260 of DNA sample after quick cooling: 0.3079
A260 of DNA sample after slow cooling: 0.2900
1.3 Isolation of Plasmid DNA and characterization by Agarose Gel Electrophoresis
Figure 2: Supercoiled plasmid DNA of E.Coli. Lane 1:
pBR322 DNA.
Lane 2: pBR322 DNA. Lane 3:
Supercoiled E.Coli plasmid DNA sample after DNA
extraction
Discussion
1.1 DNA Extraction and characterization by Agarose Gel Electrophoresis
DNA ladder is used to determine the sizes of DNAs across electrophoresis gel. This is
due to its logarithmic property between distance of fragments travelled and the sizes of
the fragments. Smearing in electrophoresis (Lane 2) might be due to excess DNA in
well. The smear at the end of Lane 4 might be due to protein contamination.
DNA will be denatured under high temperature, which the double helix will
unwind and become single-stranded. This is because the weak hydrogen bonds holding
base pairs together are broken by heat. DNA can still be renatured when the temperature
goes down to lower than melting temperature. The extent of renaturation depends on
sequence complexity and rate of reannealing. For fast cooled DNA, despite that the
DNA renatures to double helix conformation, the high rate of reannealing does not
sufficient time for the DNA base pairs to rearrange itself completely, resulting in partial
renaturation. The slow cooled DNA has sufficient time for base pairs to rearrange
themselves as complete double helix. Both the extent of denaturation and the A260 of
DNA samples in ascending order are the same, from slow cooling to fast cooling to
without cooling. The phenomenon of UV absorbance increasing as DNA is denatured
is known as the hyperchromic shift. The aromatic rings in purine and pyrimidine bases
in DNA absorb UV light strongly. Double-stranded DNA absorbs UV less strongly than
denatured DNA due to the stacking of nucleotide bases. Single stranded
deoxynucleotides with more exposed aromatic rings will thus absorb more UV than
double helix DNA.
1.2 Ultraviolet Measurement and Denaturation of Isolated DNA
Figure 3: Absorbance profiles of DNA and
protein samples from 240 to 290 nm.
(Held, 2001).
Figure 3 shows the absorbance of DNA at various wavelengths. The peak absorbance
of DNA is at 260nm, whereas protein absorbance peaks at 280nm. Thus, the
A260/A280 ratio can be an indication of purity in both nucleic acid and protein extractions.
Pure DNA and RNA preparations have expected A260/A280 ratios of >1.8 and >2.0
respectively. The A260 in this experiment is higher than A280. The A260/A280 ratio
is almost 1.8 Thus, the experiment result is justified.
There are several factors that may affect A260/A280 ratios. The 260 nm
measurements are made very near the peak of the absorbance spectrum for nucleic acids,
while the 280 nm measurement is located in a portion of the spectrum that has a very
steep slope. As a result, very small differences in the wavelength in and around 280 nm
will effect greater changes in the A260/A280 ratio than small differences at 260 nm.
Consequently, different instruments will result in slightly different A260/A280 ratios on
the same solution due to the variability of wavelength accuracy between instruments.
Individual instruments, however, should give consistent results. Concentration can also
affect the results, as dilute samples will have very little difference between the
absorbance at 260 nm and that at 280 nm. With very small differences, the detection
limit and resolution of the instrument measurements begin to become much more
significant. The type(s) of protein present in a mixture of DNA and protein can also
affect the A260/A280 ratio determination. Absorbance in the UV range of proteins is
primarily the result of aromatic ring structures. Proteins are composed of 22 different
amino acids of which only three contain aromatic side chains. Thus, the amino acid
sequence of proteins would be expected to have a tremendous influence on the ability
of a protein to absorb light at 280 nm (Held, 2001). Abnormal 260/280 ratios usually
indicate that the sample is either contaminated by protein or a reagent such as phenol
or that there was an issue with the measurement.
1.3 Isolation of Plasmid DNA and characterization by Agarose Gel Electrophoresis
Purification of plasmid DNA from bacterial DNA is based on the differential
denaturation of chromosomal and plasmid DNA, by using alkaline lysis in order to
separate the two. The basic steps of plamid isolation are disruption of the cellular
structure to create a lysate, separation of the plasmid from the chromosomal DNA, cell
debris and other insoluble material. Bacteria are lysed with a lysis buffer solution
containing sodium dodecyl sulfate (SDS) and sodium hydroxide. During this step
disruption of most cells is done, chromosomal as well as plasmid DNA are denatured
and the resulting lysate is cleared by centrifugation, filtration or magnetic clearing.
Subsequent neutralization with potassium acetate allows only the covalently closed
plasmid DNA to reanneal and to stay solubilized. Most of the chromosomal DNA and
proteins precipitate in a complex formed with potassium and SDS, which is removed
by centrifugation.
The bacteria is resuspended in a resuspension buffer and then treated by 1%
SDS (w/v) / alkaline lysis buffer to liberate the plasmid DNA from the E. coli host cells.
Neutralization buffer neutralizes the resulting lysate and creates appropriate conditions
for binding of plasmid DNA to the silica membrane column. Precipitated protein,
genomic DNA, and cell debris are then pelleted by a centrifugation step and the
supernatant is loaded onto a column. Contamination like salts, metabolites, and soluble
macromolecular cellular components are removed by simple washing with ethanolic
wash buffer. Pure plasmid DNA is finally eluted under low ionic strength conditions
with slightly alkaline buffer.
The extracted plasmid DNA is in supercoiled form; thus, it cannot be compared
to the standard pBR322 DNA, its size also cannot be measured by using a DNA ladder.
Conclusion
The A260/A280 ratio of DNA is 1.6761, around the desired A260 value of 1.8. This
indicates that the extracted chromosomal DNA is with minimum protein contamination.
DNAs experience hyperchromic shift, which the UV absorbance increases as DNA is
more denatured. This is proven by the ultraviolet measurement and denaturation of
isolated DNA experiment. The size of extracted E.Coli chromosomal DNA is measured
to be 16kb, which is almost the same as standard lambda DNA. The result shows that
the procedures of characterization of E.Coli chromosomal DNA are accurate.
References
Elkins, K.M., 2012. Forensic DNA Biology: A Laboratory Manual. [e-book]
United States: Academic Press. Available at: Google Books <books.google.com>
[Accessed 10 November 2019].
Held, P. 2001. Acid Purity Assessment using A260/280 Ratios. [online]
Available at: < https://www.biotek.com/resources/application-notes/nucleic-acidpurity-assessment-using-a260/a280-ratios/> [Accessed 10 November 2019].