Name: Wong Pui Mun
Student ID: 20WLR08576
Course code: BABS2253 Recombinant Biotechnology
Experiment : Preparation of Bacterial Plasmid DNA
Title: Isolation of Plasmid DNA by alkaline lysis method and purification of DNA.plasmids
by using nanodrop spectrophotometer.
Objectives:
1. To investigate how isolation of plasmid DNA by alkaline lysis works.
2. To determine the purity of plasmid DNA of E.coli bacteria by nanodrop
spectrophotometer.
3. To investigate the acceptable range of the ratio of the measurement absorbance that
indicates the purity of plasmid DNA.
Introduction:
Escherichia coli is the workhorse of molecular biology, serving as a factory for the synthesis
of large amounts of cloned DNA. While doing preparation of bacterial plasmid DNA, a step
where extraction of DNA must be done first. Plasmid DNA is cloned in bacteria; that is,
identical copies are made and propagated in bacteria. These bacterial cells are a complex
mixture of plasmid DNA, chromosomal DNA, protein, membranes, and cell walls.The trick
in isolating pure plasmid DNA is to separate it from the rest of the cell components and from
chromosomal DNA. The most common method used for separating plasmid DNA from
chromosomal DNA is the alkaline lysis method. The developer of this technique exploited the
supercoiled nature of plasmid DNA to separate it from chromosomal DNA (Casali & Preston
2003). In the alkaline lysis method, both chromosomal and plasmid DNA are denatured by
NaOH, which disrupts the hydrogen bonding. Denatured DNA can reanneal at neutral pH if it
is not kept in alkaline conditions for too long. The two halves of the plasmid double-helix
DNA remain intertwined during the incubation in alkali and they are in close proximity for
reannealing (Casali & Preston 2003). Chromosomal DNA is much larger, not interwinted,
and often fragmented. Chromosomal DNA remains denatured and is precipitated by
potassium acetate and sodium dodecyl sulfate (SDS), an ionic detergent. The precipitated
chromosomal DNA is usually removed by filtration or centrifugation. RNA is also generally
removed during the alkaline lysis step simply by adding RNase to the buffer. Plasmid DNA
remaining in the supernatant can then be precipitated by ethanol. Beside, the plasmid DNA
can be then further purified over an anion exchange column.
Anion exchange chromatography works by taking advantage of the negatively charged
nature of DNA where A matrix of positively charged molecules is immobilized in the column
(Kieleczawa 2006). When the cellular components are applied to the column, the negatively
charged nucleic acids absorb to the positively charged matrix. Proteins and other undesirable
cellular debris are washed out of the column, and only the nucleic acids remain. The highly
purified plasmid DNA can then be eluted from column using a high-salt buffer (Kieleczawa
2006). DNA quantification is accomplished by reading the absorbance of a known volume of
sample at 260 nm. The absorption coefficient of pure DNA is 50 𝞵g/ml (Matlook B 2015).
This means that one A260 unit of double stranded DNA corresponds to 50 𝞵g DNA per ml.
To assess the purity of a DNA sample, the ratio of the absorbance at 260nm over the
absorbance at 280 nm is calculated. A ratio of approximately 1.8 is ideal. A sample with a
higher ratio may have RNA contamination, and a sample with a lower ratio may have protein
contamination. However, in this experiment, anion exchange chromatography did not
accomplish.
Hypothesis:
Pure DNA typically yields a 260/280 ratio of 1.8 to 2.0 and a 260/280 ratio of 2.0 to 2.4 for
DNA and RNA, respectively.
Procedure:
4 empty tubes were prepared with each tube filled with 3 ml of E coli bacteria. 4 of the tubes
were centrifuged with the speed of 10,000 rpm for about 30 seconds per pellet. After
centrifuge, the pellet and the supernatant were obtained. The pellet was then resuspended in
100 μl of GTE solution and was let to sit for 5 minutes at room temperature while the
supernatant was thrown away. The pellet with the mixture of GTE solution was ensured to
mix thoroughly by gently tapping the tubes with fingers. Then, 200 μl of NaOH and SDS
solution were added to each tube and were mixed evenly. The tubes were then allowed to sit
for 5 minutes at room temperature. After that, 300 μl of potassium acetate solution was
pipetted and added into the mixture in each tube. The tubes with solution mixture were
inverted up and down to make sure that the mixtures were mixed thoroughly. Next, the tubes
were put into the ice box for 5 minutes. After 5 minutes, the tubes were centrifuge with the
speed of 10000 rpm for 3 minutes to pellet cell debris and chromosomal DNA. Next, the 400
μl of supernatant was transferred to another 4 new tubes. 95% ethanol was added to each of
the tubes and was mixed thoroughly and were allowed to sit for 1 minutes. Later, the tubes
were centrifuge again with the speed of 10000 rpm for 1 minutes to pellet plasmid DNA and
RNA. The supernatant was removed. 70% of ethanol solution was added to each tube
respectively and the step of centrifuge with 10000 rpm was repeated for 1 minutes. The
supernatant was removed and the cap of the tubes were left open for 1 minutes to let excess
ethanol to evaporate. Next, the pellet of each tube was resuspended with 30 μl of TE buffer
with pH 8 and was mixed thoroughly. Moreover, 1 μl of TE buffer with pH 8 was pipetted
and was put onto the plate of the nanodrop spectrophotometer and this is the blank. The
concentration of each sample was then measured by the nanodrop spectrophotometer. After
that, the 1 μl of sample of each tube was pipetted onto the plate respectively. The graph and
the concentration of each tube was observed and recorded.
Results:
Table 1 The concentration of the sample in each tube and their A260/280 and A260/230.
Tube Concentratio A260 A280 A260/280 A260/230
n of the
sample
(ng/μl)
1 79.7 1.595 0.797 2.00 4.26
2 155.2 3.104 1.579 1.97 2.86
3 86.0 1.721 0.868 1.98 4.38
4 152.4 3.048 1.547 1.97 2.54
Discussion:
The purity of DNA can be assessed using various methods including absorbance, agarose
gel electrophoresis, or use of fluorescent DNA-binding dyes. All three methods are
convenient, but have varying requirements in terms of equipment needed, ease of use and
calculations to consider. In this experiment, the most common techniques were used to
determine the DNA yield and purity which is by the measurement of absorbance through
nanodrop spectrophotometer at 260nm compared to the value measured at 280nm.
To accurately assess sample quality, 260/280 or 260/230 ratios should be analyzed in
combination with overall spectral quality. The ratio of absorbance at 260nm 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 while a ratio of 2.0 is generally accepted as pure 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 280nm. There is a second measurement of nucleic acid purity where it is
used to indicate the presence of unwanted organic contaminants such as trizol, phenol,
guanidine HCl and guanidine thiocyanate. This measurement of absorbance at 260nm and
230nm (A260/230). Expected 260/230 values are commonly in the rage of 2.0 to 2.4. If the
ratio is out of the general acceptable range, it may indicate the presence of contaminants
which absorb at 230nm. This ratio is actually dependent on the pH and ionic strength of the
buffer used to make the blank and sample measurements. Acidic solutions will
under-represent the ratio by 0.2-0.3, while a basic solution will over-represent the ratio by
0.2-0.3 (Koetsier & Cantor 2019).
In the case of contaminants by phenol or trizol extraction, residual reagent contamination
may be indicated by abnormal spectra between 220 to 240 nm as well as by shifts in the 260
to 280 nm region; conversely, residual guanidine from column extraction may contribute to a
peak near 230 nm and a shift in the trough from 230 nm to approximately 240 nm (Wilfinger,
Mackey & Chomczynski 1997). In this experiment, the results shown in table 1 could
obviously be seen that the ratio of absorbance at 260nm and 280nm and the ratio of
absorbance at 260nm and 230nm are in the acceptable range for 4 of the tubes. DNA
concentration is estimated by measuring the absorbance at 260 nm, adjusting the A260
measurement for turbidity, multiplying by the dilution factor and using the relationship that
an A260 of 1.0 = 50µg/ml pure dsDNA (Matlook B 2015). Based on table 1, the
concentration of the sample was calculated in nanogram/microliter, it is the same with the
unit of microgram/mL. Total yield of DNA is obtained by multiplying the DNA
concentration by the final total purified sample volume. As the total purified sample volume
was not recorded during the experiment, therefore the DNA yield (ng) was not calculated.
Both purity ratios are unusable below concentrations of 20 ng/µl, and are still relatively
variable (with a tendency to be too high) at values up to 50 ng/µl. In this experiment, the
concentration of four of the tubes are higher than 20 ng/µl and so two of the ratios of
absorbance are considered. All the ratios of A260/280 shown in table 1 are in acceptable
range for four of the tubes meaning that there is no contamination occurring in the samples.
However the 260/230 ratios of four of the tubes are higher than the expected range. This may
be because the operator made a blank measurement on a dirty pedestal or using an
inappropriate solution for the Blank measurement (Matlook B 2015).
Furthermore, there are some troubles that will cause abnormal 260/280 ratios. First is that
samples are too dilute and concentrations are adjacent to the lower detection limit. As
mentioned above, nucleic acid samples with concentration that are less than 20ng/µl will
cause an abnormal ratio and the result should not be considered. Besides, an appropriate
solution is very important to be used for the blank solution where the properties of ionic
strength and pH of the solution used should be similar with the sample solution. As the
solution used will directly influence the ratio of the absorbance.
Conclusion:
In conclusion, the hypothesis stated that pure DNA generally has a ratio of A260/280
between 1.8 to 2.0 and A260/230 ratio between 2.0 to 2.4. A ratio that is lower than the
expected ratio range would indicate that there is contamination of the sample such that the
contaminants may be trizol, phenol and residual guanidine. In this experiment, the A260/280
ratio of all the tubes are in acceptable range that indicates no chemical residuals left in the
sample however the A260/230 ratios is higher than expected because of the human error.
References:
1. Casali, N. & Preston, A. 2003 E. coli plasmid vectors: methods and applications,
Springer Science & Business Media, United Kingdom.
2. Kieleczawa, J. 2006, DNA sequencing II: optimizing preparation and cleanup, Jones
& Bartlett Learning, Sudbury, Massachusetts
3. Koetsier, G. & Cantor, E. 2019, A practical guide to analyzing nucleic acid
concentration and purity with microvolume spectrophotometers, New England
Biolabs, Ipswich, England.
4. Matlook, B. 2015, Assessment of nucleic acid purity, Thermo Fisher Scientific,
Wilmington, United States.
5. Wilfinger, 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.
