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POLYMERASE CHAIN REACTION

(First Week)
Rüveyda AKÇİN, Gebze Technical University, Turkey

AIM
The amplification of the pac 3,5 gene, isolated for the purpose of cloning from Escherichia coli.

INTRODUCTION
PCR is process of amplifying nucleic acids under  7 µl dH2O
appropriate conditions. It is in vitro and in vivo  2.5 µl 10x PCR buffer w/o MgCl2 (1x)
method. Sufficient quantities can obtain from
very small samples. PCR is used in many (It is used to facilitate the binding of primers.
diseases, diagnosis of infections and They usually include: Tris-HCI (10-50 mM), ≥
microorganisms. Also, it has advanced 1,5 mM MgCl, ≤ 50 mM KCI)
evolutionary biology.
 5 µl MgCI2 25 mM (5 mM)
There are three stages of PCR. The first stage is
(MgCl is necessary to binding Taq polymerase
Denaturation. DNA to be used is denatured at
to dNTPs, primers and template DNA. It
94-98  C for 20-30 seconds at high
provides polymerase activity.)
temperature. The second stage is Annealing.
The primers added at 50-65  C are ligated to  2.5 µl dNTP 2 mM (200 µM)
the target site. Finally stage is Extension. DNA
(It should be used at equal concentration from
polymerase enzyme provides primer extension
each dNTP to minimize the falses. dNTP is
with dNTPs at 72  C.
selected according to size of DNA product and
There are some points to note in PCR. These the number of PCR loops. Generally, it puts
ones; enzyme selection and concentration, after from buffer.)
dNTPs, concentration of MgCl2 or Mg2SO4 and
 2 µl Forward and 2 µl Reverse primer (40
primers. (They will be mentioned in the
ng/µl) (80 ng)
material part.)
(Primers determine success of PCR studies.
In PCR, the binding temperature is determined
Primers should harmonious with target region.
by calculating the Tm value of each primer.
Length of primers is important. If possible,
Applicable binding temperature is below 1-2
palindromic primers should be selected.)
 C of Tm temperature of primers.
 2 µl Template DNA 100 ng/ µl (200 ng)
Tm= (4x[G+C]) +(2x[A+T])  C
 2 µl Taq DNA polymerase 0.45 U/µl (0.9 U)
There are many types of PCR such as Multiplex
PCR, Broad-range/Consensus PCR, Nested PCR, (The characteristic of this enzyme is
In-situ PCR, Random Amplified Polymeric DNA, thermostability. Also, it can be polymerized 37-
Reverse Transcriptase. 100 nucleotides.)

PROTOCOL
MATERIALS
1. All materials except DNA Taq polymerase
are put in an eppendorf.
2. Place the tubes in a thermal cycler 72  C 210 seconds
preheated to 94  C. 5. Perform a final extension step at 72  C for
3. Hold the tubes at 94  C for 5 minutes for 10 minutes.
the first denaturation step, then add the 6. Store the amplified DNA at 4  C for short
polymerase. term storage or at -20  C for long term
4. Perform the following temperature cycles storage. (It's done to complete all the DNA.)
for 30 times: 7. Visualise the 2 µl of the amplified DNA via
94  C 60 seconds agarose gel electrophoresis.
64  C 60 seconds

RESTRICTION ENZYME DIGESTION


(Second Week)

AIM
Cutting of pAc 3.5 gene with HindIII and EcoRI from amplified DNA for cloning.

INTRODUCTION
Restriction enzymes break sugar and Nomenclature
phosphate backbone of DNA, its double-
The first three letters of the name are italicized
stranded recognition sequence. In 1968, K.W.
because they abbreviate the genus and species
Wilcox, H.O.Smith and T.S. Kelley was first
names of the organism. The fourth letter
isolated from Haemophilus influenzae bacteria.
comes from the bacterial strain designation.
The recognition sequences are same in both
Roman numerals are used to identify specific
DNA chains for some restriction enzymes.
enzymes from bacteria that contain multiple
These are called palindiromic sequences.
restriction enzymes. Roman numeral indicates
Restriction enzymes have two functional
order in which restriction enzymes were
subunit. These are DNA recognition regions
discovered in a particular strain.
and catalytic domain. DNA recognition region
occurs from N-terminal region formed Type I
approximately 390 monomers. N terminal
Type I restriction enzymes have three subunits
region occurs subunits D1, D2, D3. Restriction
called HsdR, HsdM and HsdS. These enzymes
enzymes recognize specific recognition site and
cut distinct regions at least 1000 base pairs
place the catalytic domain here. The catalytic
from the recognition sites. Adenosine
site which is placed in DNA recognition region
triphosphate (ATP) and magnesium ions (Mg 2+)
breaks phosphodiester bonds in helix.
are required for the activity of enzymes.
Restriction endonucleases are used in many
Type II
areas. For instance, in creating a DNA map,
population polymorphism analysis, They have a dimer structure formed by a single
preparation of probes, forming of mutant type of protein. Recognition regions are
organisms, analysis of the modification states polindromic. They know DNA and cut that
of DNA. region. In general, they is used only Mg2+ as
cofactor.
Restriction endonucleases are categorized into
three or four general groups.
Type III (Ethyl alcohol and sodium acetate are used
to completely remove water from the DNA.
They cut DNA away from recognition site 20-30
Also, they are used to stop reaction of
bases. Also, they have many subunits. They
restriction enzyme and to precipitate DNA
need AdoMet and ATP for methylation and
thus volume is reduced. )
restiriction of DNA.

Star Activity: In non-optimal conditions,  1 µl RNase DNase-free (1/2 diluted)


specificity of enzyme to recognition sequence
(For pUC19 gene because pUC19 is not a
changes. This called star activity.
PCR product and It needs to be used to
MATERİALS remove RNA.)

 39 µl dH2O PROTOCOL
 5 µl Buffer B (10x) 1. All materials are put in an eppendorf.
2. Incubate the tubes at 37  C for 90 min.
(Common buffer for both HindIII and EcoRI. 3. Add 1 µl RNase DNAase-free (1/2 diluted)
It is necessary for optimal pH adjustment and incubat the tubes at 37  C for
for enzyme function.) additional 15 min. (This stage for pUC19.)
4. Add 5 µl sodium acetate 3 M (pH 5.2) and
 2 µl pUC19 plasmid (2 µg) or pAc 3.5 gene 125 µl volumes of 100 % ethanol.
(1 µg) 5. Chill the tubes at -20  C for 2 h or -80  C
for 30 min.
(pAc 3.5 is PCR product. Normally, pAc has 6. Centrifuge in a microfuge (+4 C) at 14.000
2.6 kb but we added 900 base to increase rpm for 15 min.
the activity of the enzyme. This plasmid is 7. Remove the supernatant and add 100 µl of
used in this experiment. pUC19 encodes for 70-75 % ethanol.
an ampicillin resistance gene. pUC19 is 8. Centrifuge in a microfuge (+4 C) at 14.000
small but has a high copy number. ) rpm for 5 min.
9. Remove the ethanol and dry the pellet
 2 µl HindIII 4 U/ µl dilution (4 U/ µg) and 2
in a centrifugal evaporator for 10-20
µl EcoRI 4 U/ µl dilution (4 U/ µg)
min.
(EcoRI enzyme cuts from recognition 10. Resuspend the pellet in 10 µl dH2O.
sequences of circular DNA (5 '...GAATTC ... 11. Add 1 µl of 6x gel loading dye.
3') (3 '... CTTAAG ... 5' ) and creates two 12. Load the digested DNA sample in a 1 %
adhesives end. HindIII cuts from agarose gel and electrophorese at 80 volts
(5’...AAGCTT....3’) (3’…TTCGAA…5’)) until dye markers have migrated an
appropriate distance (about 1 hour).
 5 µl NaAc (0.3 M pH 5.2) + 125 µl EtOH 13. Apply the “DNA Isolaion from Agarose Gel”
%100 protocol.

AGAROSE GEL ELECTROPHORESIS


(Third Week)
AIM
pUC19 plasmid and pAc 3.5 gene cut by restriction enzymes walk on agarose gel and isolation of 3 kb
areas.

INTRODUCTION
The net electric charges that molecules possess  50x TAE (242 g Tris base, 57.1 ml glacial
have an effect on the movement of these acetic acid, 37.2 g Na2EDTA.2H2O, H2O to 1
molecules in an electrical field. The l, pH  8.5)
electrophoresis technique is based on this
principle. Agarose gel electrophoresis is used (The buffer maintains pH and ion balance
for cleavage, purification and identification of of gel. The buffer gives ions to solution for
nucleic acid fragments. The agarose gel electrical conductivity. The same buffer
samples are walking usually run in horizontal should be used in the gel preparation and
position, with constant power and electrical in the tank.)
field. Agarose is obtained from seaweed and it
is an unbranched chain polymer. The agarose is  6x Loading dye (Bromophenol Blue 0.15 %,
insoluble in the buffer at room temperature. It Sucrose/Glycerol 30 %)
dissolves in boiling water. Defines DNA and
RNA molecules between 200 and 50,000 bp (Bromophenol blue, because it carries a
dimensions. The most effective agarose slight negative charge at neutral pH values,
concentrations for the separation of nucleic it runs in the same direction with DNA and
acids are 0.3-2.0 %. proteins in the gel. Sucrose/Glycerol gives
the sample density and markes them
remain in the wells.)

 RedSafe Nucleic Acid Stainning Solution


(20.000X)

(It enters the broken structure of DNA and


gives radiation. So that, DNA bands give
radiation in the gel.)
Table 1. (Apporopriate agarose concentrations
for separating DNA fragments of various sizes.) PROTOCOL
There are three factors that affect migration (Preparation of 1% agarose gel because to
rate through a gel: looked to 3 kb band.)
1. Molecular size of DNA 1. To prepare 30 ml of a 1 % agarose
2. Agarose Concentration
solution, measure 0.3 g agarose
3. Conformation of DNA
into a glass flask.
4. Applied voltage
2. Microwave for 1-3 min (until the
5. Base composition and voltage
6. Presence of intercalating agents agarose is completely dissolved).
7. Electrophoresis buffer composition 3. Let agarose solution cool down
(50-55  C ) for about 5 min.
MATERİALS 4. Add 3 µl of 20.000x RedSafe
 Agarose (elecrophoresis grade) Nucleic Acid Staining Solution to the
agarose solution. Swirl the flask
gently to mix the solution and avoid (There are a total of 50 μl samples. 50
forming bubbles. µl divided into two wells.) and pUC19
5. Pour the agarose solution into a gel (30 µl divided into three wells.)were
tray with the well comb in place. combined. Namely, There are 4 wells in
total and a well marker.
6. Place newly poured gel at 4  C for
9. Electophorese at 100 volts until dye
10-15 minutes OR let sit at room
markers have migrated an appropriate
temperature for 20-30 minutes, until it
distance, depending on the size of DNA
has completely solidified.
to be visualized.
7. To run, gently remove the comb, place
10. Visualise and analyze the gel under UV
tray in the electrophoresis buffer (the
light using a transilluminator. DNA can
same buffer used to prepare the
be visualised under short wave UV light
agarose).
if the DNA will not be used further; or
8. To prepare samples for
with a longwave UV light if the DNA is
electrophoresis, add 1 µl of 6x gel
to be cut out and purified.
loading dye for every 5 µl of DNA
solution. Mix well. In this experiment,
pAc 3.5 plasmid was dissolved in 10 μl
of water. Then 5 groups of pAc 3.5

DNA ISOLATION FROM AGAROSE GEL

AIM
Cutting of the 3 kb band of pAC 3.5 and pUC19 walked in the agarose.

INTRODUCTION
The NucleoSpin kit was used in this experiment. MATERİALS
Buffer NE (5 mM Tris/HCl, pH 8.5), buffer NT3
and buffer NTI are used in this kit.  1.5 ml microcentrifuge tubes
Contaminations are removed by a simple  Disposable ppette tips
washing step with ethanolic Buffer NT3. For gel  Scalpel
extraction the agarose gel slice is dissolved in
PROTOCOL
high-salt Buffer NT. Pure DNA is finally eluted
under low ionic strength conditions with 1. A total of five eppendorfs were named NTI,
slightly alkaline Buffer NE (5 mM Tris/HCl, pH NT3, Elution buffer and pAC 3.5 (2).
8.5). Binding Buffer NTI with pH indicator. 2. Excise the DNA fragment from the agarose
gel with a clean, sharp scalpel and transfer
it into a microcentrifuge tube.
3. Empty weight of eppendorf is 1 gr. The
weight of second band in the gel is 0.059
grams. That’s why place 120 μl NTI (0.059
 0.060, 0.060 x 2 =120 μl)
4. Incubate sample at 50  C for 5-10 min (or
until the gel slice has completely
dissolved). To help dissolve gel, mix by
vortexing the tube every 2-3 mi during the
incubation.
5. Place a NucleoSpin Gel and PCR Clean-up
Column into a provided 2 ml collection
tube and load up to 700 μl sample.
6. Centrifuge for 30 s at 11.000x g. Discard
flow-through and place the column back
into the same collection tube.
7. Load remaining sample if necessary and
repeat the centrifugation step.
8. To wash silica membrane, add 700 μl Buffer
NT3 to the NucleoSpin Gel and PCR Clean-
up Column and centifuge forr 30 s at
11.000x g.
9. Discard flow-through and place the column
Figure 1.
back into the same collection tube.
10. Centrifuge for 1 min 1t 11.000x g to Figure 1 shows the result of made PCR isolation
remove Buffer NT3 completely. Make sure from after DNA isolation. Agarose was done to
the spn column does not come in contact see the PCR result. Single band is observed in
with the flow-through while removing it groups 3. , 5. and 9.
from the centrifuge and the collection
tube.
11. Place the NucleoSpin Gel and PCR Clean-up
Column into a new 1.5 ml microcenrifuge
tube.
12. To elute DNA, add 15-30 μl of Buffer NE (It
is used to precipitate DNA.) or H2O to the
center of the silica membrane in
NucleoSpin Gel and PCR Clean-up Column
and incubate at room temperature (18-25
 C) for 1 min.
13. Cenrifuge the column for 1 min at 11.000x
g.
14. Quantify the eluted DNA by using
nanodrop spectrophotometer and
visualise it in a 1 % agarose gel.
15. Use appropriate amount of purified DNA in
molecular cloning experiment and store Figure 2.
the remaining DNA at -20  C.
Figure 2 is result of agarose of pAc 3.5 gene and
RESULT pUC 3.5 plasmid cut with restriction enzymes.
The bands are visible in 3 kb area.
CONCLUSIONS https://www.slideshare.net/agaroz-jel-
elektroforezi-ki-boyutlu-jel-elektroforez
In general, purpose of these experiments are
cloning pAc 3.5 gene isolated from Escherichia http://yunus.hacettepe.edu.tr/~coner/GEN/0
coli. Firstly, PCR was performed after DNA 1/rest.htm
isolation, because It is necessary to reproduce
http://www.biyologlar.com/agaroz-jel-
a single genomic DNA obtained for steps. The
elektroforezi
amount of MgCI2 used in PCR is different for
each group. In this way, An ideal 2 mM http://biyokure.org/restriksiyon-enzimleri-ile-
concentration of MgCI2 should be observed. On kesim/142/
the other hand, in Figure 1, the results are not
http://www.mn-
very good. Only the 3rd, 5th and 9th wells have
net.com/Products/DNAandRNApurification/Cl
very weak bands. Also, many dNTPs are not
eanup/NucleoSpinGelandPCRCleanup/tabid/1
used. That could be the reason, In the previous
452/language/en-US/Default.aspx
experiment A (260/280)→1.74, A
(260/230)→1.34 results were obtained for
group 5 because of there is a risk of
contamination.

Secondly, EcoRI and HindIII were used as


restriction enzymes, because both the pUC19
plasmid and the pAc 3.5 gene must cut. They
were cut with the same enzymes to make they
compatible. Agarose is made to look at the
correctness of the cut parts and to determine
the kb to be isolated. This is result in Figure 2.
(Before loading into the wells, centrifugation
was done several times to reduce volume.)
Despite use of RNase, there are still RNA
residues. RNase may be caused by not being
fully active. Also, carefully, the bands of pUC19
and pAc 3.5 appear to be in different kb,
because pAc is normally 2.6 kb. However, we
added 900 base to increase the activity of the
enzyme.

Finally, The 3 kb portions was isolated with the


NucleoSpin kit for next experiment.

REFERENCE
http://bys.trakya.edu.tr/file/open/44259122

http://biyokure.org/pcr

http://yunus.hacettepe.edu.tr/elektroforez.pd
f