Universiti Teknologi MARA Cawangan Perak

Kampus Tapah

Faculty of Applied Sciences

Diploma in Science

BIO301

Practical 2:
Techniques in DNA Technology

Lecturer:
DR Low Kim Fatt

Group:
A4AS1205_A

Student name:
NORATIKAH BT MOHAMAD ARIFIN

Student ID:
2017184257
Practical 2.1: Plasmid DNA isolation using alkaline lysis method
Introduction
Alkaline lysis was first described by Birnboim and Doly in 1979 which has a few
modifications and has been the preferred method for plasmid DNA extraction from bacteria. Alkaline
lysis is a method used in molecular biology, to isolate plasmid DNA or other cell components such as
proteins by breaking the cells open. It is probably one of the most generally useful techniques
because it is a fast, reliable and relatively clean way to obtain DNA from cells. The purified plasmid
DNA can be used for immediate use in all molecular biology procedures such as digestion with
restriction enzymes, cloning, PCR, transfection, in vitro translation, blotting and sequencing.
Purification of plasmid DNA from bacterial DNA is based on the differential denaturation of
chromosomal and plasmid DNA 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.
The principle of the alkaline lysis method is a kind of magic. After suspending the bacteria
culture in the solvent which is solution I, an alkaline solution which is solution II is added to the
sample. In this condition, almost all proteins are denatured. DNA double-strand structure is also
denatured to single-strand. Even though this condition is quiet extreme, supercoiled plasmid DNA
still remains its stable structure and do not denatured. After 5 minutes, incubation of alkaline
denature, high-salt buffer which know as solution III is added for the purpose of a sudden change of
pH in the solution. As a result, denatured protein and chromosomal DNA do not turn back to its own
structure, causing these molecules insoluble. On the other hand, plasmid DNA remains soluble, thus
centrifuge step easily separates the plasmid DNA from debris of proteins and chromosomal DNA.
The isolation of plasmid DNA from bacteria is a crucial technique in molecular biology and is an
essential step in many procedures such as cloning, DNA sequencing, transfection, and gene therapy.
Therefore, these manipulations require the isolation of high purity plasmid DNA. The objective of
the experiment is to extract and isolate a plasmid from a bacterial and to expose the student with the
application of centrifuge.

Methodology
Materials and apparatus
o Bacteria culture (Escherichia coli culture)
o 1.5 mL Microcentrifuge tubes
o Centrifuge
o Micropipette and tips (1000µL, 200µL, 20µL)
o Incubator shaker
o -20°C freezer
o Glucose
o Tris-Cl
 o Ethylene diamine tetraacetic acid (EDTA)
o Sodium hydroxide (NaOH)
o Sodium dodecyl sulphate (SDS)
o Potassium acetate
o Glacial acetic acid
o sdH2O
o Ethanol 96% v/v, 70%v/v (cold)
o Nutrient broth media
o Dry ice or ice cubes
o Tris/Borate/EDTA (TBE)
o RNase
o Solution I (Glucose 50mM; Tris-Cl 25mM; EDTA 10mM)
o Solution II (NaOH 0.2N; SDS 1%w/v)
o Solution III (Potassium acetate 5M, glacial acetic acid)

Procedure
A. Preparation of E.coli Culture
1. A day before this practical, bacterial culture was inoculated in 100 mL of nutrient broth.
2. The culture was incubated for about 8-24 hours, at 200rpm and 35°C.
B. Plasmid extraction
1. Gently shake the bacteria culture in the shake flasks and transfer 1 mL to a clean and sterile
microcentrifuge tube. Label the microcentrifuge tube as A.
2. Spin the microcentrifuge tube at 10000rpm by using a centrifuge for 1 minute.
3. Drain the supernatant (media broth) and leave the pellet (bacteria) in the microcentrifuge tube.
4. Wash the pellet with 1 mL sdH2O by gently mixing the culture by tapping the microcentrifuge
tube.
5. Again, spin the microcentrifuge at 10000rpm for 1 minute, the drain the supernatant and leave
the pellet.
6. Gently resuspend the pellet with 200µL of solution I and leave for 5 minutes at room
temperature.
7. Next, add the mixture with 400µL of solution II, inverse gently and put it in ice for 5 minutes.
8. After 5 minutes, add 300µL of solution III, inverse gently and put in ice for 5 minutes.
 9. Spin the mixture in microcentrifuge tube at 12000rpm for 11 minutes.
10. Carefully transfer the supernatant of the mixture in microcentrifuge A into a new sterile
microcentrifuge tube, without disturbing the pellet. Label this microcentrifuge tube as A1.
11. Discard the A microcentrifuge tube into a disposal plastic bag. Use A1 microcentrifuge tube
for further analysis.
12. Add double volume of 96% ethanol to the A1 microcentrifuge tube.
13. Next, keep the A1 microcentrifuge tube in -20 °C for about 10 minutes.
14. Then, spin the microcentrifuge tube A1 at 12000rpm for 10 minutes.
15. Draw off the supernatant from the tube. Add with 500µL of 70% cold ethanol into the tube.
16. Spin the A1 microcentrifuge tube at 12000rpm for 5 minutes.
17. Gently discard the supernatant from the A1 tube.
18. Open the tube cap and place the tube in the laminar air-flow for drying.
19. After the tube is completely dried, add 20µL of sdH2O and 1µL of RNAase, mix it gently in
the tube.
20. Keep safely the tube in -20 °C for about one week to preserve the plasmid for electrophoresis.

Expected results
Bacteria containing the plasmid of interest is first grown, and then allowed to lyse with an
alkaline lysis buffer consisting of a detergent sodium dodecyl sulfate (SDS) and a strong base sodium
hydroxide. The detergent cleaves the phospholipid bilayer of membrane and the alkali denatures the
proteins which are involved in maintaining the structure of the cell membrane. Through a series of
steps involving agitation, precipitation, centrifugation, and the removal of supernatant, cellular debris
is removed and the plasmid is isolated and purified.

Figure 1: First stage of plasmid DNA isolation using alkaline lysis method.
 The first stage of the mini-prep involves bursting the cells using an alkaline solution. This
releases their contents into the surrounding liquid. An acidic solution is then added, which neutralizes
the alkaline solution and denatures the proteins, causing them to become insoluble. They can be
removed from the cell lysate through centrifugation (Figure 1).

Figure 2: Second stage of plasmid DNA isolation using alkaline lysis method.
The second stage of the mini-prep involves passing the cell lysate through a column. These
columns bind onto plasmid DNA, and allow chromosomal DNA and other cell products to pass
through. After washing the column several times, the column release the plasmid DNA by passing
through an elution solution. Thus, the plasmid is isolated and purified (Figure 2).
Plasmids exist in a complex topologically intertwined supercoiled structure inside the cell.
Due to the mechanical or shear stress applied during extraction, the plasmid comes in some other
forms like open circular, linear, covalently bounded (figure 3). But, the supercoiled plasmids are
mostly accepted by cells during transformation or transfection processes. The final plasmid should be
free from any traces of contaminants like degraded RNA, genomic DNA, bacterial proteins and
endotoxins.
Figure 3: Different contaminants and isoforms of plasmids after plasmid extraction process.
Photograph obtained after 1% agarose gel electrophoresis with voltage of 5 volt/cm of gel for 1.5
hours.

Practical 2.2: Agarose gel electrophoresis

Introduction
Electrophoresis is a process to identify, separate, and characterize the biological molecules
like DNA, RNA, or proteins. There are two type of electrophoresis which is vertical gel
electrophoresis and horizontal gel electrophoresis. Vertical gel electrophoresis runs samples from
upside down and discontinuously. It is most suitable for protein separation. For example, PAGE-
polyacrylamide gel electrophoresis is a type of vertical gel electrophoresis. Horizontal gel
electrophoresis runs samples continuously, parallel to the surface and it is widely used for separating
DNA. Instead of polyacrylamide, agarose is used in horizontal gel electrophoresis. Horizontal gel
electrophoresis is easier, more reliable and gives the best results. However, for the separation of the
molecule with a smaller difference, PAGE is more reliable in comparison with agarose gel
electrophoresis. In this practical agarose gel electrophoresis is used for DNA separation. The agarose
gel electrophoresis is also known as submarine gel electrophoresis because the entire gel remains
covered with the running buffer, completely.
Therefore, the principle of gel electrophoresis is DNA molecules which is negatively charged
migrate towards the positive charge under the influence of constant current. If put into an electric
field, it will move from the negative pole to the positive pole. A gel serves as the matrix for the
movement of the DNA molecule. The gel is a semi solid material with certain size pores. If DNA is
cut into fragments of different sizes, during electrophoresis the smaller ones are able to move faster
through the pores of the gel than the larger ones. This creates a pattern where the fragments are
arranged according to their sizes from the negative to the positive pole. Moreover, there are three
factors that affect migration rate through a gel which is size of the DNA, conformation of the DNA,
and the last one is ionic strength of the running buffer. However, while doing agarose gel
electrophoresis, the bands are either very light or not visible despite the amount of good DNA quality
and quantity. The objective for this experiment is to separate DNA fragments, to observe DNA
fragment and to estimate the size of DNA fragments.
Methodology
Material and apparatus
o Plasmid extract (from practical 2.1)
o Micropipette and tips (1000µL, 200µL, 20µL)
o Gel electrophoresis set and power supply
o UV transilluminator
o Hot plate
o Gel safe stain
o xl Tris/Borate/EDTA(TBE)
o RNAase
o Dry ice or ice cubes
o Loading dye
o DNA marker 1 kb
o Agarose powder

Procedure
1. Prepare an agarose gel by using dissolved 0.35 Agarose powder and boil it in 50 mL xl TBE
until it is crystal clear.
2. Add 5µL of gel safe stain into the dissolved gel. Swirl gently.
3. Set the rack and the comb. Pour the gel into the rack to solidify the gel accordingly.
4. After waiting for about 30 minutes, remove the comb from the gel gently, put the gel into the
case and fill in the case with xl TBE until the maximum level.
5. Take out the microcentrifuge tube, A1 from the -20°C and thaw for a few minutes. Prior
loading to the gel, add 1µL of RNAase into the tube A1 and mix it gently.
6. Transfer 10µL from the A1 tube and add it with about 2µL of loading dye.
7. To load the DNA marker, transfer 10µL from the marker tube and add it with about 2µL of
loading dye.
8. Load the sample and the DNA marker into the well gently.
9. Close the lid of the electrophoresis chamber and apply current 100 V for 30 minutes with 15
mL of gel with position from negative to positive current.
10. Visualize the DNA bands with the UV transilluminator, take photo of the gel.
11. Compare the estimate the DNA band samples by using the DNA marker band.
Expected results
Agarose gel electrophoresis is an easy way to separate DNA fragments by their sizes and
visualize them. It is a common diagnostic procedure used in molecular biological labs. The technique
of electrophoresis is based on the fact that DNA is negatively charged at neutral pH due to its
phosphate backbone. For this reason, when an electrical potential is placed on the DNA it will move
toward the positive pole. The plasmid was found in many different supercoiled forms in the bacteria.
When plasmid was isolated from a bacterial culture, in this case, E. coli all the different supercoiled
forms of the plasmid was isolated, and each will migrate differently on the gel, producing about three
major bands and many minor bands. When this mixture of supercoiled plasmids is cut with a
restriction enzyme, the different forms linearize and unwind. As a result they all become identical
and run at the same rate, and usually only one band seen on the gel.

Figure 4: Black-and-white photograph of an agarose gel.

The staining agent Ethidium Bromide was applied to gel and the buffer solution to visualise
the DNA fragments. The DNA fragments in the samples 1 to 11 moved from their origin which is the
sample slots through the gel towards the positive electrode from top to bottom in the picture. The gel
matrix acts as a sieve. The smaller DNA molecules migrate faster than larger ones, so DNA
molecules of different sizes separate into distinct bands during electrophoresis. More DNA in a band
gives more intense staining of that band. For example, 50ng of DNA in a band gives two times
brighter staining than 25ng. This can be seen in lane 7 in (Figure 4) where restriction fragments
originating from one microgram of identical DNA molecules are separated which means the bands
contain equimolar amount of DNA. From the figure above it show that the smallest fragment of 564
basepairs (1) is hardly visible, while the biggest fragment of more than 23.000 basepairs (2) shows a
very bright band. However, Band 3 contains the smaller DNA fragments compare to band 2, but it
still much brighter. This is because there is more nanograms of DNA in 3 than in 2 which is the
number of molecules in 3 must be much higher than in 2.