Plasmid Isolation and Gel Electrophoresis

Shannen Krishna S. Sena

AB-2L

November 06, 2017

A scientific paper submitted in partial fulfillment of the requirements in Principles of Ecology

laboratory under Prof. Aimee Lynn A. Barrion-Dupo, 2nd sem., 2015-2016.

 ABSTRACT

Plasmid isolation was performed via the alkaline lysis method. The
strains used were Escherichia coli BW20767/pRL27 and E. coli with
pGEM, which are kanamycin resistant and ampicillin resistant,
respectively. An E. coli wildtype was used to serve as the control setup.
The strains were added with three solutions (I, II and III) which aid for
the lysis and release of the cellular constituents involving the plasmid
DNA. A series of centrifugation was done to pellet out the plasmids.
Also, the concentration and purity of the extracted DNA was verified
spectrophotometrically while the structure and size of the plasmid was
determined via agarose electrophoresis. From the results of the
electrophoresis, the size of the plasmids obtained was 0.1 kb, which was
relatively smaller to the theoretical size of the strain that is 4kb (E. coli
BW20767/pRL27) and 3kb (E. coli with pGEM). The plasmids gathered
may be smaller than the theoretical size of the plasmid or the DNA
sample was already fragmented.

INTRODUCTION

After World War II, the study on plasmids arose as antibacterial drugs were starting to be

commonly used. Scientific laboratories have isolated bacteria with antibiotic resistance and they have

noticed the rapid spread of the resistance (Summers, 2009). The resistance was then associated to the

presence of plasmids in the bacteria. Furthermore, plasmids have been significant to the development of

molecular biotechnology because they serve as vectors for gene transfer between bacteria. They have

been widely distributed among bacteria due to their own origin of replication that enables them to

replicate independently from the bacterial chromosome. Also, since plasmids carry various types of genes

that help the bacteria to adapt to adverse conditions, the bacteria containing plasmids are more likely to

pass it to daughter cells or to other bacteria. Such genes involve those responsible for antibiotic

resistance, ultraviolet-C (UV-C) resistance, unusual carbon sources catabolism and heavy metal

detoxifying enzymes (Banu and Prasad, 2017). They can be transferred via conjugation, transformation

and transduction. Most plasmids are covalently-closed circular double-stranded DNA molecules but

linear plasmids have also been observed in Borrelia and Streptomyces (Barbour, 1988; Holmes and

Jobling, 1996). Their size range is from 1 kb to 200 kb (Schumman, 2008). The criteria for classifying
plasmids involves their conjugative ability or mobilizability, incompatibility groups, copy number and

host range (Schumman, 2008,).

Depending on their properties, plasmids are classified as F-plasmid, Col plasmid, R plasmid,

Suicide plasmid and Virulence plasmid (Banu and Prasad, 2017). In this experiment, R plasmids or R

factors, which contains genes for antibiotic resistance, are used (Holmes and Jobling, 1996). Specifically,

the strains used were a kanamycin resistant Escherichia coli BW20767 /pRL27 and an ampicillin resistant

E.coli with pGEM.

In plasmid isolation, alkaline lysis method is usually performed. This is a method in which

alkaline solutions are used to remove cell debris and lyse the cells, thereby, releasing the plasmid DNA

(Ehrt and Schnappinger, 2003). In addition, gel electrophoresis is executed to determine the size and

structure of the plasmid. The electric mobility of the DNA sample would depend on the size of the DNA.

Smaller plasmid DNA would migrate faster through the gel as compared to those that have larger sizes.

In the experiment, the same methods were used. The isolation of plasmid was done via the

alkaline lysis method. Agarose gel electrophoresis was performed to determine the size and structure of

the plasmid and then a BioRad Gel documentation system was used to further visualize the plasmid DNA.

For this experiment, it is hypothesized that plasmids could be isolated via the alkaline lysis procedure.

Moreover, the purity and concentration of the plasmid can be confirmed spectrophotometrically while

their size and structure can be determined electrophoretically.

The experiment aimed to isolate the plasmid of E. coli BW20767/pRL27 and E. coli with pGEM.

The specific objectives were to:

1. isolate the plasmid DNA via alkaline lysis procedure

2. determine the purity and concentration of the extracted DNA spectrophotometrically

3. determine the plasmid size and structure through agarose gel electrophoresis
 The study was conducted at Room C-321, Research Laboratory and Molecular Biology and

Biotechnology Laboratory, Institute of Biological Sciences, Biological Sciences Building of the College

of Arts and Sciences, University of the Philippines Los Banos, Laguna from October 24-26, 2017.

MATERIALS AND METHODS

Plasmid Isolation

Plasmid isolation was performed by initially growing Escherichia coli BW20767 /pRL27 (kanamycin

resistant) and E. coli with pGEM (ampicillin resistant), separately into 30 ml LB broth with 50 ug/ml

kanamycin and 100 ug/ml ampicillin, respectively. For the control, E. coli wildtype was grown in a 30 ml

LB broth without antibiotics. The broths were incubated at 30 C for 24 hr.

After incubation, 1 ml of each strain were transferred to a sterile eppendorf tube in which 5 tubes

were allotted for the E. coli BW20767/pRL27, 5 tubes for E. coli with pGEM and 2 tubes for the E. coli

wildtype. The cells on the tube were pelleted out via centrifugation at 10,000 rpm for 2 minutes. The

supernatant was removed prior to the resuspension of the cells in 100 ul of Solution I. The solution

contained 50 mM glucose, 10 mM ethyldiaminetetra-acetic acid (EDTA), 25 mM Tris-HCl (pH 8.0) and

2 mg/ml of lysozyme that was added before use. The EDTA was added to allow binding of cations, such

as Mg 2+, to allow lipid molecules in the lipopolysaccharide to pack more compactly, thereby,

destabilizing the outer membrane of the cell. Moreover, Tris served as a pH buffer while the lysozyme

was to digest the cell wall. The suspension was vortexed vigorously to ensure that the pellets were

completely dissolved. A 200 ul of Solution II (pH 12.45), which contained 0.2 N NaOH and 1% sodium

dodecyl sulfate (SDS), was added to the previous suspension. SDS was responsible for the release of

cellular components, including the DNA, via solubilizing the membrane. It was also added at high pH to

release fats by chemically breaking down lipids. The resulting suspension were then mixed gently on a

vortex before it was kept on ice for 5 minutes. Once the suspension appeared clear and viscous, 150 ul of

Solution III (pH 4.8), containing 3 M Na acetate, 1X Tris Acetate EDTA (TAE) buffer and Tris EDTA

(TE) buffer (pH 8.0), was added to neutralize the suspension. The TAE buffer was prepared by mixing
4.48 g Tris base, 1.142 ml glacial acetic acid and 2 ml of 0.5 M EDTA. Moreover, the TE buffer was

prepared with 10 mM Tris-HCl and 1 mM EDTA. The suspension was neutralized by Solution III to

allow renaturation of the circular plasmid DNA to become double-stranded DNA. The resulting

suspension of the three solutions containing the cells were mixed gently via inversions before it was kept

on ice for 5 minutes. It was again centrifuged at 12, 000 rpm for 5 minutes to separate the supernatant into

a second sterile eppendorf tube. Cold ethanol with a volume of 1 ml was added to the supernatant to

precipitate the plasmid DNA. The solution was mixed via inversion before it was kept on ice for 5

minutes. The plasmid DNA was then pelleted out via another centrifugation at 12,000 rpm for 5 min at

4C. The ethanol was discarded while the tubes containing the pellets were inverted and air dried.

After drying, the DNA was dissolved in 30 ul of TE buffer to solubilize the DNA while protecting it

from degradation. The plasmid DNA concentration was determined via NanoDrop spectrophotometry.

Also, the purity and the OD reading at 260 and 280 nm were obtained.

Gel Electrophoresis

Before running subjecting the DNA sample to electrophoresis, the plate and the end spacers of

the setup were first washed and rinsed with ethanol to remove the grease. Moreover, 40 ul of 1.0-1.5%

agarose in 0.5X TAE was prepared and boiled until the agarose was completely dissolved. Upon cooling

at about 50 C, GelRed was added and then the agarose solution was poured on the gel plate. GelRed was

used because it gives off a red fluorescence under UV light. The spacers were then inserted into the plate

and the gel was allowed to solidify. The spacers created the wells for the loading of the sample.

Furthermore, 2 ul of the DNA sample was added with 2 ul of loading buffer to increase the density of the

sample and to allow it to sink upon loading into the wells. After the gel solidified, the spacers were slowly

removed and the gel was set in the mupid in such a way that the samples moved from the negative to the

positive electrode. The gel was submerged below 2-3mm of the buffer.

The setup used 12 lanes in which lane 1 was loaded with 1 ul of the molecular weight marker,

lanes 2-6 with 4 ul of E.coli with pGEM DNA sample, lanes 7-11 with 4 ul of E. coli BW20767 /pRL27
DNA sample and lane 12 with the E. coli wildtype. The apparatus was ran at 50 V for 30 minutes until the

tracking dye has travelled two-thirds of the gel length.

After electrophoresis, the gel was inserted in a BioRad gel documentation system to obtain

images of the bands.

RESULTS AND DISCUSSION

After the procedures, the results from the spectrophotometer confirmed the purity of the extracted

DNA. A 1.8 ratio of the OD reading at 260 nm and 280 nm was considered a pure DNA (Wilfinger et.al.,

1997). Nucleic acids are absorbed at 260 nm while proteins are absorbed at 280 nm. It indicates that if the

DNA sample is pure without proteins, then the absorbance at 260 nm is high, thereby, resulting to a high

ratio (approximately 1.8) between the absorbance at 260 nm and 280 nm. From the data in Table 1, the

sample located on D2 obtained a 1.8 ratio, thus, the purity of the samples were confirmed. Moreover, the

concentration of the DNA sample was also indicated in the data.

Table 1. NanoDrop Spectrophotometry results of the extracted DNA from E. coli BW20767 /pRL27 and E. coli with
pGEM.

Sample Location Name 260 280 320 260 280 260/280 ng/ul

Read# Raw Raw Raw

1 C2 2.023 1.083 0.166 1.879 0.927 2.028 1878.843
1 C3 2.985 1.998 0.159 2.864 1.864 1.536 2863.627
1 D2 2.146 1.268 0.221 1.944 1.056 1.841 1944.015
1 D3 2.917 1.573 0.183 2.774 1.41 1.9688 2773.941
1 E2 1.858 0.939 0.08 1.796 0.867 2.073 1796.481
1 E3 3.117 2.144 0.322 2.841 1.853 1.534 2841.331
Yellow background indicates a high reference wavelength read

Consequently, the DNA sample were subjected to electrophoresis to determine the size and

structure of the sample. In Figure 1, the image of the electrophoresis results were shown. Lane 1 contains

the molecular weight marker, Lanes 2-6 contains E. coli with pGEM, Lanes 7-11 contains E. coli

BW20767 /pRL27 and Lane 12 contains the E. coli wildtype. The topmost band on the ladder was 12 kb
while the bottom was 100 bp or 0.1 kb. The plasmid for E. coli with pGEM and E. coli BW20767 /pRL27

has a size of 3kb (Vector pGEM-T: Information, n.d.) and 4kb (Althubiani, 2013), respectively. In an

electrophoretic apparatus, plasmids tend to migrate faster in the gel due to their compact size. DNA

molecules with a smaller size tend to travel through the setup faster as compared to those with a larger

size size. However, considering the theoretical size of plasmids of the strains used, the bands of the

sample should be located in between the top and bottom part of the ladder. As indicated in Figure 2, the

bands of the DNA sample extracted were located at the bottom strand of the marker, which was at 0.1 kb.

This could possibly mean that the DNA sample obtained were fragmented because of the smaller

kilobases indicated by the results or the plasmid obtained was smaller than the theoretical size of the

plasmids of the strains used.

Figure 1. Photo of the results of the agarose gel electrophoresis in which lane 1 - molecular weight marker, lanes 2
to 6 - E. coli with pGEM, lanes 7 to 11 - E. coli BW20767 /pRL27 and lane 12 - E. coli wildtype

SUMMARY AND CONCLUSION

 Plasmids are covalently-closed circular double stranded DNA which carry genes that are non-

essential to the bacteria but it helps them survive through adverse conditions. An example of those genes

is antibiotic resistance. In the experiment, two strains containing plasmids coding for antibiotic resistance

were used. The strains were E. coli BW20767 /pRL27 and E. coli with pGEM, which are kanamycin

resistant and ampicillin resistant, respectively. Moreover, an E. coli wildtype was used for the control.

The plasmids of the strains were isolated via alkaline lysis procedure methods. The cellular constituents

of the bacteria used were released via a series of centrifugation and addition of solutions that would lyse

the cells. There were three solutions used for the lysis of the cells. Solution I involved the addition of

EDTA, which was responsible for destabilizing the outer membrane of the cell, Tris as the pH buffer, and

lysozyme which aid in digesting the cell wall. Solution II contained SDS which caused the release of

cellular components, including the DNA, via solubilizing the membrane of the cell. It also released fats

when added at high pH. Lastly, Solution III was added to allow the renaturation of the plasmid into

double stranded DNA. Furthermore, a volume of LB broth inoculated with the strain underwent a series

of centrifugation before and after the addition of the three solution to pellet out the released cellular

constituents. Cold ethanol was also used to precipitate the plasmid DNA. The pellet obtained after the

final centrifugation was air dried and then it was mixed with TE buffer to solubilize the DNA while

protecting it from degradation. The sample was further subjected to spectrophotometry for the

confirmation of purity and then it was followed by electrophoresis to determine the size and structure of

the plasmid.

Theoretically the plasmid size of E. coli BW20767 /pRL27 and E. coli with pGEM were 4kb and

3kb, respectively. Due to the compact and small size of the plasmids, they tend to migrate faster through

the apparatus. However, the results from the electrophoresis showed that the size of the samples were 0.1

kb. The obtained plasmids were smaller than the theoretical size of the plasmid of the strain or the

obtained samples were fragmented.

 In conclusion, plasmid DNA can be isolated via the alkaline lysis procedure. The purity and the

concentration of the extracted DNA could be determined spectrophotometrically. Moreover, performing

electrophoresis could confirm the isolation of the plasmid by comparing the actual sized obtained from

the theoretical size of the plasmid strains used.

LITERATURE CITED

Althubiani, A. (2013). Characterisation of an Antimicrobial Producer Ioslated from the Surface of

Seaweed. doi: 10.13140/RG.2.2.25164.21122.
Barbour, A. G. (1988). Plasmid analysis of Borrelia burgdorferi, the Lyme disease agent. Journal of
Clinical Microbiology, 26(3), 475–478.
Banu, H. and Prasad, K.P. (2017) Role of Plasmids in Microbiology. Journal of Aquaculture Research &
Development 8, 466. doi:10.4172/2155-9546.1000466
Ehrt, S. and Schnappinger, D. (2003). Isolation of Plasmids from E. coli by Alkaline Lysis. In N. Casali
and A. Prestion (Eds.), E. coli Plasmid Vector: Methods in Molecular Biology, Volumer 235, pp.
328. New Jersey, USA: Humana Press.
Holmes, R.K., and Jobling, M.G. (1996). Genetics. In S. Baron (Ed.), Medical Microbiology (4th ed.) (pp.
87-138). Texas: University of Texas Medical Branch, Department of Microbiology.
Schumman, W. (2008). Escherichia coli Cloning and Expression Vectors. In G. Lipps (Ed.), Plasmids:
Current research and future trends (pp. 1-19). Great Britain: Caister Academic Press.
Summers, D.K. (2009). The Biology of Plasmids. USA: Blackwell Publishing Ltd. pp. 1-19.
Vector pGEM-T: Information and Usage Suggestion (Plasmid map only for reference). (n.d.) Retrieved
from http://www.sinobiological.com/Vector-pGEM-T-a-1636.html on November 4, 2017.
Wilfinger, W.W., Mackey, K. and Chomczynski, P. (1997). Effect of pH and Ionic Strength on the
Spectrophotometric Assessment of Nucleic Acid Purity. BioTechniques, 22, 474-481