Molecular biology of the cell with Lab

Instructor: Assem Duisembekova

Section #1

Week #2

Title: Plasmid extraction and DNA analysis

Name: Alibek Ysmaiyl

Introduction
Plasmid is a circular DNA molecule which is found in many bacterial cells as two stranded
double helix. Actually, the term plasmid was used by Joshua Lederberg in 1952 in order two
explain that plasmid is a separate chromosomal piece of genetic information which can be passed
between generations. As the plasmid is located separately from the chromosome it is known that
plasmid participates in conjugation in which the plasmids are transferred between different
organisms resulting in genetic transfer of DNA. Moreover, plasmids are beneficial for bacteria
by different aspects especially antibiotic resistance genes which allow bacteria to survive in
environment containing the antibiotic such as ampicillin. In science, the plasmids are also known
as vectors when the study is performed to clone, transfer, and so one. These plasmid vectors can
be obtained by a process called plasmid extraction or isolation [1].

There are many ways to extract the plasmid DNA from bacterial cells. First of all, bacterial
cells are grown in growth medium for plasmid extraction. When a certain detergent treatment is
used in opening the bacterial cell plasmid extraction begins. The obtained solution after opening
the bacterial cell knows as lysate is centrifuged. Liquid supernatant which contains the plasmid
DNA is extracted from tube and kept in order to avoid the DNA denaturation [1].

The general method of this experiment is alkaline lysis method. This method consists of cell
growth and harvesting, resuspension, cell lysis, neutralization, and then clean-up DNA sample.
Buffer holding Tris and EDTA is utilized in order to resuspend the cells [2]. Divalent cations
such as Ca2+ and Mg2+ are involved in the prevention of plasmid damage by DNases stabilizing
the bacterial membrane of E.coli, and EDTA helps in binding between them destabilizing the
membrane [3]. After that, a solution of sodium hydroxide and Sodium Dodecyl Sulfate (SDS) is
added making the membranes more soluble leading to bacterial cell lysis process. After 5 min
incubation, cell lysis is neutralized by Potassium acetate which is in fact cause the renaturation
of DNA from ssDNA to dsDNA due to hydrogen bonding. Finally, after centrifugation, the pure
plasmid DNA is eluted by small amount of elution buffer in order to stabilize and store for a long
period [3].

The next step is gel electrophoresis in which molecules are separated via a porous semisolid
matrix (gel from agarose and buffered water) due to the different rates of agility in an involved
electric field. In fact, molecules move through the agarose gel according to their length, charge,
and sizes of gel pores [2]. So, in general spectrophotometer and gel electrophoresis are
performed for DNA analysis throughout the laboratory experiment.

The purpose of this experiment is to extract the plasmid DNA from E.coli bacterial cells which
contain pGLO gene for GFP and analyze the plasmid DNA for its purity.

Materials and procedure

Materials: ice bucket, microfuge tubes, Aurum plasmid mini kit, ethanol, potassium acetate,
agarose gel, centrifuge machine, pipettes.
Procedure

E.coli with pGLO was grown in LB+Amp growth medium and then bacterial culture
concentration was calculated by utilizing the spectrophotometer and adding 50 l of
bacterial culture to 950 l growth medium at =600 nm.

After OD calculation, required volume of bacterial culture which is 5 ml was placed into
capped microfuge tube, and then the cells were centrifuged for 1 minute. All supernatant
was removed by pipetting.

250 l of resuspension solution was added and then the cell pellet was resuspended.

250 l of lysis solution was added and capped tube was inverted for 6-8 times until
solution became viscous and slightly clear.

After 3 minutes, 350 l of neutralization solution was added and inverted for 6-8 times
until the visible precipitation was formed.

Then, neutralized lysate was centrifuged for 5 minutes, and plasmid mini column was
prepared.

750 l of wash solution was added plasmid mini column and centrifuged for 1 minute.

Finally, plasmid mini column was transferred into microfuge tube and after 50 l of
solution was added onto the membrane stack at the base of the column and left for 1
minute for saturation, plasmid mini column was centrifuged for 1 minute in order to elute
the plasmid.

After that, DNA was quantified by using the NanoDrop and analyzed running 1% agarose
gel. That is, DNA loading dye was transferred to sample, and then pipetted into certain
wall. Then, agarose gel was run at 80-150 V, and after gel electrophoresis, gel was
analyzed under UV-trans illuminator.

Results
Calculation:

(OD600 of undiluted culture)* x (culture volume in ml) = #ODml

*OD600 of 1.0 = 8 x 10 8 cells/ml

(OD 600 of undiluted culture)

Culture volume in ml = OD ml

Since, 0 absorbance was observed for our groups sample, it is advised to take 5 l of culture
sample.
Picture 1.Observation on gel electrophoresis

Our groups sample was loaded to the 5th wall, so it can be seen that there is no run of bands on
the gel.

Picture 2.DNA concentration by NanoDrop spectrophotometer.

Our groups sample concentration which is under the name of ALIBEK was 94.73 ng/ l.

Discussion
According to results, it cannot be considered that the laboratory experiment was performed
successfully, because important gel electrophoresis part results were not obtained. Due to picture
1 the 5th wall was loaded by sample, but after gel run, there were no bands at all not to mention
the fact that GFP was not observed under UV light. However, before gel electrophoresis,
concentration of sample was calculated and found to be 94.73 ng/ l. As it was instructed, there
are two outcomes of this result, namely, high concentration of plasmid DNA or impurities were
present. In general, this result demonstrates that there were DNA strands in the sample, however,
it is so unusual that gel electrophoresis did not show any bands at all. Moreover, when OD was
measured, the absorbance was zero, and it was suggested by instructor that bacterial culture
concentration was too high, so that the spectrophotometer could not calculate the absorbance.
However, other groups have taken spectrophotometer results around 1.75-1.84 absorbance.
Finally, it is thought that there was a gross error in which some of our group member mixed up
tube containing the plasmid DNA with tube containing water before loading the gel
electrophoresis.

Taking all points into consideration, even if the experiment was not completed successfully,
the key points of plasmid DNA isolation process was understood, and performed in practice. The
results cannot be considered as reliable because of mentioned gross error or spectrophotometer
could not determine the concentration.

Reference list
1. McConnell, M. B. Plasmids https://www.coursehero.com/file/8276185/Plasmids/ (accessed Feb 5,
2017).

2. C. B. G. isolation of e. coli chromosomal dna lab report - Isolation of E. Coli

https://www.coursehero.com/file/6245560/isolation-of-e-coli-chromosomal-dna-lab-report/ (accessed Feb
5, 2017).

3. Duisembekova, A. Plasmid extraction and DNA analysis. Lab 3 presentation.