Experiment-I: Isolation of PCV1 and Confirmation by

Restriction Digestion
Introduction
What is Plasmid?
A plasmid is an extra chromosomal, super coiled DNA and capable of independent
replication. They have their own origin of replication. They use most of the host cells’ own
DNA replication enzymes (such as DNA polymerases, ligases, primases) in order to make
copies of themselves. In many cases, it is circular and double-stranded. On agarose gel DNA
migrates according to its molecular weight, but plasmids have different forms and each form
migrates differently. Supercoiled DNA migrates faster than linear form of the same plasmid
and linear plasmid migrates faster than circular nicked from of the same plasmid.
Plasmids are found in bacteria and sometimes in eukaryotes. There are different types of
plasmids like F (Fertility plasmids) and R (Resistance plasmids). Plasmids carry one or more
genes and often these genes give the host cells an ability to survive under unusual environmental
conditions. So, plasmids are important for bacterial adaptation. For example, the host cells can
survive in the presence of antibiotics like ampicillin and several heavy metals by the help of
plasmids that carry genes, which provide resistance to ampicillin or heavy metals.
In molecular biology, plasmids are used as molecular engineering tools. They are used to
clone parts of DNA (complete sequence or a part of gene, regulatory sequences etc) to mostly
express RNA, protein or investigate functions of non-coding sequences.
In this laboratory section, you will isolate pCV-1 plasmid. It is a mammalian expression
vector containing TAT (trans-activator of transcription) and Rev (transactivating protein) genes
of HIV. Total size of the plasmid is 10.3. PstI enzyme cuts this plasmid from two different sites
and gives rise to two fragments of 7.0 kb, the plasmid backbone, and 3.3 kb, insert including
1.8 kb cDNA of TAT and Rev and 1.5 kb of adjacent pBR322 plasmid sequence.
Isolation principle of plasmid DNA
All methods developed for plasmid isolation contain 4 main stages:
1) Growing a culture of the bacteria
2) Harvesting and lysis of the bacteria
3) Removal of all the components except the plasmid DNA
4) Purification of the plasmid DNA.
Firstly, the host cell containing plasmid of interest is grown overnight in a medium (LB)
with a suitable antibiotic. Then, bacterial culture is harvested by spinning the culture in a
centrifuge and finally bacterial cells are disrupted by different methods; chemical or physical.
Physical methods involve disruption of the cells by mechanical forces. Chemical methods,
which are more commonly used, include chemical agents that disrupt the integrity of the cell
barriers. Lysozyme and EDTA, which are used separately or in combination, are commonly
used chemicals for chemical digestion. Lysozyme digests the cell wall, while EDTA removes
magnesium and calcium ions that are essential for preserving the overall structure of the cell
envelope. SDS, which is another commonly used chemical, is a detergent and helps the process
of lysis by removing lipid molecules from cell membranes. Chemical lysis can also include use

11
of a strong alkali reagent that denatures the host cell chromosomal DNA by disrupting base
pairing. However, this alkaline reagent does not denature plasmid DNA due to its compact
structure.
DNA Concentration Determination
It is extremely important to know the exact concentration of DNA in a solution for the
following experiments such as gene cloning or transfection. Nucleotide concentrations can be
measured by UV absorbance spectrophotometry. Specifically for DNA, absorbance at 260 nm
is directly proportional to the concentration of DNA. Another parameter that can be measured
by UV absorbance is purity of the DNA solution. The ratio of absorbance at 260 nm to 280 nm
(A260/A280) is an indicator of purity that 1.8 indicates a pure DNA sample. Ratio higher than 1.8
indicates RNA contamination, while ratio lower than 1.8 means sample is contaminated with
protein or phenol. The ratio of absorbance (A260/A230) is an indicator of alcohol contamination
that a value lower than 1.8 indicates to a contaminated sample.
What is restriction enzyme?
Restriction enzymes are part of a bacterial defense mechanism, where bacterial enzymes
digest the DNA of infecting phage viruses. Bacterial DNA, on the other hand, is protected
against these enzymes by methylation. These enzymes that recognize a specific sequence and
then cleave DNA at specific positions are called restriction enzymes. Recognition sequences
are usually palindromic, which means that the sequence reads the same forward (5’ to 3’ on one
strand) as backward (3’ to 5’ on the complementary strand) (Table 1.1). Recognition sequence
is usually six nucleotide long but for some enzymes it can be shorter, such as four, or longer
such as eight or more. Depending on where the enzyme cuts, restriction reaction can generate
sticky (cohesive) or blunt end DNA fragments. The enzymes that cut from the same position
on both strands generate blunt ends (SmaI in Table 1.1). The enzymes that cut at different
positions on the two strands generate sticky or cohesive ends (PstI in Table 1.1), which can
stick the two end together due to base pairing.
Table 1.1 Examples of restriction enzymes

Cleavage
Enzyme Recognition Sequence After cleavage Type

PstI Sticky
5’- 5’-
…CTGCAG…
5’- -3’
…CTGCAG…
5’- -3’
…CTGCAG…
…CTGCAG… -3’
-3’ 5’-
5’- …CTGCA
…C…C
5’-
5’- …C TGCAG…G…-3’
TGCAG…
TGCAG… -3’
-3’
-3’
3’- 3’-
…GACGTC…
3’- -5’
…GACGTC…
3’- -5’
…GACGTC…
…GACGTC… -5’
-5’ 3’-
3’- …G
…GACGT
3’-
3’- …GACGT
…GACGT ACGTC…
C…C… -5’
-5’
C… -5’
-5’

SmaI Blunt
5’- …CCCGGG… -3’
5’- 5’- …CCCGGG…
5’- -3’
…CCCGGG…
…CCCGGG… -3’ -3’
5’-
5’-
5’-
5’- …CCC
…CCC
…CCC
…CCC
GGG…
GGG…
GGG…
GGG…
-3’
-3’-3’
-3’
3’-
3’-
3’- …GGGCCC…
…GGGCCC…
…GGGCCC… -5’
-5’
-5’ 3’-
3’- …GGG
…GGG
3’- …GGG CCC…
CCC… -5’
-5’
CCC… -5’
3’- …GGGCCC… -5’ 3’- …GGG CCC… -5’

A restriction reaction contains the DNA as substrate, appropriate amount of restriction

enzyme (1 unit of enzyme cuts 1 µg of DNA in 1 hour), appropriate enzyme buffer that provides
optimum conditions (ioning strength, PH etc) for the enzyme activity. Although most enzymes
work best at 37 °C, some has highest activity at different temperatures such as 50 or 75 °C.

5’- 5’-
5’- CCC
CCC GGG
CCC GGG -3’ -3’
GGG -3’
3’-
12
5’- 3’-
CCC 3’- GGG
GGG CCCCCC
GGGGGG 5’-- 5’
--3’
CCC 5’

3’- GGG CCC - 5’

What is agarose gel electrophoresis?
Agarose gel electrophoresis is a technique used to separate DNA molecules primarily based
on their molecular weight. At neutral pH, double-stranded DNA has a uniform negative charge
due to phosphate group. Therefore, in an electrical field DNA fragments migrate from the
cathode (-) towards the anode (+). In agarose electrophoresis, DNA molecules migrate in an
electric field through a molecular matrix composed of the seaweed extract, agarose.
Although the migration of DNA through an agarose gel is mainly a function of the size of
the molecule, there are several other factors that affect the migration of DNA within an agarose
gel. These include:
1) Conformation of DNA affects its migration pattern. DNA fragments at the same size
but in supercoiled, relaxed, linear, circular or nicked circular forms migrate different
from each other.
2) Agarose concentration of the gel affects the migration of DNA. Agarose forms a
porous 3D structure when polymerized, and DNA passes through those pores during
migration. Smaller pore sizes, obtained by higher agarose concentration, will retard
the migration; while larger pore sizes, obtained by lower agarose concentration, will
facilitate migration of even larger fragments.
3) The intercalating dyes, such as positively charged ethidium bromide (EtBr) are used
to visualize the DNA fragments on agarose gel. Binding of these dyes affects
migration mainly by changing charge and conformation of DNA.
4) Electrical conductivity, which is determined by the composition of the buffer,
increases mobility of DNA in agarose gel. However, increased electrical conductivity
also causes over heating of the system, which results in melting of the agarose gel.
Tris acetate – EDTA (TAE) buffer is the most commonly used buffer in agorose gel
electrophoresis.
Intercalating dyes such as ethidium bromide (EtBr) are used to visualize DNA bands in
agarose gels under UV light. Caution: ethidium bromide is a strong mutagen. Always wear
gloves while handling gels, solutions or samples containing ethidium bromide.
DNA samples are mixed with a loading dye before being loaded into the wells of the agarose
gel. Loading dye contains glycerol, which is required for the DNA samples to sink into wells
and prevent spill over. Loading dye also contains at least one visible dye to estimate how much
DNA migrated. Bromophenol Blue and xylene cyanol, which migrate approximately at 400bp
and at 4kb respectively in 1% agarose gel, are the most commonly used dyes. Loading of a
DNA ladder, which is a mixture of DNA fragments with known sizes, provides a more accurate
estimate of the size of the DNA sample.

13
Materials and Methods
PCV-1 plasmid
pCV-1 is a mammalian expression vector containing TAT (trans-activator of transcription)
and Rev (transactivating protein) genes of HIV. Total size of the plasmid is 10.3. It contains
tetracycline resistance gene. PstI enzyme cuts this plasmid from two different sites and gives
rise to two fragments of 7.0 kb, the plasmid backbone, and 3.3 kb, insert including 1.8 kb cDNA
of TAT and Rev and 1.5 kb of adjacent pBR322 plasmid sequence.
Buffers and Solutions
LB Medium (Luria-Bertani Medium)
1 Liter:
Add the following components to 950 ml deionized H2O.
Tryptone 10 g
Yeast extract 5 g
NaCl 10 g
Shake the mix until everything dissolves well. Adjust the pH to 7.0 with 5 N NaOH (~ 0.2
ml). Adjust the volume of the solution to 1 liter with deionized H2O. Sterilize by autoclaving
for 15 min at 15 psi (1.05 kg/cm2) on liquid cycle.
Tetracyclin
Stock solution is 12.5 mg/ml. Prepare the working solution at the concentration of 10μg/ml.
Alkaline Lysis Buffer I
50 mM Glucose
25 mM Tris-Cl (pH 8.0)
10 mM EDTA (pH 8.0)
Autoclave for 15 minutes at 15 psi (1.05 kg/cm2) on liquid cycle. Store at +4oC.
Alkaline Lysis Buffer II
0.2 N NaOH
1% (w/v) SDS
Prepare fresh before each use and keep at room temperature.
Alkaline Lysis Buffer III
5 M potassium acetate 60.0 ml
Glacial acetic acid 11.5 ml
H2O 28.5 ml
Store the solution at +4oC. Transfer to ice just before use.

14
TAE Bufer
Working Solution Stock Solution / Liter
1X 50X
40 nM Tris-acetate 242 g of Tris-base
1 mM EDTA 57.1 ml of glacial acetic acid
100 ml of 0.5 M EDTA (pH 8.0)

Ethidium Bromide (10 mg/ml)

Dissolve 1 g of Ethidium Bromide in 100 ml H2O. Store in dark. Caution: ethidium bromide
is a strong mutagen. Always wear gloves while handling gels, solutions or samples containing
ethidium bromide.
6X Gel Loading Dye
0.25% (w/v) bromophenol blue
0.25% (w/v) xylene cyanol FF
15% glycerol in H2O
NaOH (10N)
Dissolve 4 g NaOH in 8 ml H2O, and complete the volume to 10 ml. This is a highly
exothermic reaction. Prepare the solution on ice and in plastic beakers.
%10 SDS
Dissolve 1 g SDS in 8 ml H2O and complete the volume to 10 ml with H2O. Caution: Always
wear mask when working with SDS powder.

Safety
1. While handling any experimental staff (pipette, Erlenmeyer, eppendorf, etc) always be
aware of wearing gloves.
2. Ethidium bromide is a mutagen and always wear gloves while handling all staffs possibly
contaminated with ethidium bromide.
3. Phenol:chloroform are dangerous chemicals and never inhale them and always wear
gloves while working them.
4. Never touch and use any staff in the lab without permission and information from your
lab assistants.
5. Agarose boiled in microwave can easily boil up to overflow the flask. Swirl gently with
caution.

15
Protocol
Procedure for Plasmid DNA isolation (Conventional Protocol)
1. Inoculate 2 ml of LB medium containing the appropriate antibiotic (10 μg/ml of
tetracycline) with a single colony of bacteria transformed with pCV-1. Incubate the culture
overnight at 37oC with vigorous shaking.
2. Transfer 1.5 ml of the culture into a microfuge tube. Centrifuge at maximum speed for 30
seconds at +4oC.
3. Remove the supernatant by aspiration or pipetting. Remove as much supernatant as
possible without disturbing the pellet.
4. Resuspend the pellet in 100 μl of ice-cold Alkaline Lysis Solution I by vigorous vortexing.
(Lysosome might be required to lyse rigid bacterial cell walls.)
5. Add 200 μl of freshly prepared Alkaline Lysis Solution II. Close the tube tightly, and mix
by inverting the tubes gently 5-6 times. Do not vortex. Store the solution on ice.
6. Add 150 μl of ice-cold Alkaline Lysis Solution III. Close the tube tightly and mix
thoroughly by inverting the tube several times. Store the tube on ice for 3-5 minutes.
7. Centrifuge the bacterial lysate at maximum speed for 5 minutes at +4oC in a microfuge.
Transfer the supernatant to a fresh tube.
8. (Optional) Add an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1). Mix the
organic aqueous phases by vortexing and then centrifuge the emulsion at maximum speed for
2 minutes at +4oC in a microfuge. Transfer the aqueous phase upper layer to a fresh tube.
9. Precipitate nucleic acids from the supernatant by adding 2 volumes of ethanol at room
temperature. Mix the solution by vortexing and then allow the mixture to stand for 2 minutes at
room temperature.
10. Collect the precipitated nucleic acids by centrifugation at maximum speed for 5 minutes
at +4oC in a microfuge.
11. Remove the supernatant by gentle aspiration or pipetting. Remove all drops on the walls
of the eppendorf by pipetting or tissue paper.
12. Add 1 ml of 70% ethanol to the pellet and invert the closed tube several times. Recover
the DNA by centrifugation at maximum speed for 2 minutes at +4oC in a microfuge.
13. Remove supernatant and make sure to remove any drops on the eppendorf wall. Keep
the open tube at room temperature until all liquid is evaporated (5-10 minutes).
14. Option I: Dissolve the nucleic acids in 50 μl of TE buffer (pH 8.0) containing 20 μg/ml
DNase-free RNase A. Vortex the solution for a few seconds.
Option II: Dissolve the pellet in 50 μl H2O containing 20 μg/ml DNase-free RNase A.
15. Store the DNA solution at -20oC
NOTE: If RNase A is not involved in the final step, the DNA prep will be contaminated
with RNA. Contaminating RNA is usually visible on the agarose gel, but it does not interfere

16
with the subsequent reactions such as restriction digestion. RNA contamination prevents an
accurate measurement of the DNA prep.
Restriction Digestion by PstI
In order to calculate the necessary amount of enzyme, lambda DNA, which is approximately
48.5 kb, will be used as a reference. 1 unit of enzyme cuts a single enzyme site on 1 μg lambda
DNA in 1 hour.
pCV-1 plasmid is 10.3 kb in length and has 2 PstI sites. So;
Lambda DNA: 1μg 1u 1h 48.5 kb 1 site
PCV1 DNA: 1 μg X u 1h 10.3 kb 2 sites
X = (48.5 * 2) / 10.3 u
X = 9.42 u or ~10 u PstI enzyme.
Reaction:
Calculate a 20 μl reaction:
10x Enzyme Buffer 2 μl
100x BSA (if required) 0.2 μl
PstI 1 μl (10 u)
Plasmid DNA x μl (2 μl for 1 μg/ μl)
H2O complete to 20 μl
1. Adding H2O to the reaction tube.
2. Add the enzyme buffer, BSA (if required).
3. Add plasmid DNA.
4. Mix thoroughly. (quick spin should follow in case of if you vortex the mix)
5. Add PstI enzyme.
6. Mix thoroughly by pipetting.
7. Incubate at 37oC for 5 -10 minutes.
Agarose Gel Preparation
1. While preparing agarose gel always work under the hood!
2. Assemble the tray for pouring gel. Make sure the comb fits to the tray. Place the tray
under the fume hood.
3. Add 50 ml 1X TAE into an Erlenmeyer flask and 0.5 g Agarose powder.
4. Heat the mixture in a microwave until agarose dissolved completely and the mixture
is crystal clear.
5. Cool the mixture to approximately 50 °C.

17
 6. Place the flask under the fume hood and add 0.5 μl ethidium bromide (stock
concentration is 10 mg/ml).
7. Mix thoroughly and pour the mixture to the tray.
8. Place the comb.
9. Wait until gel solidifies.
Preparation of DNA for Agarose Gel
1. Mix 5 μl of digestion mix (or 5 μl of plasmid DNA) with 1 μl of 6X loading dye.
2. Mix thoroughly by pipetting.
Loading Samples to Agarose Gel
1. Put the solidified agarose gel into the running tank.
2. Pour 1X TAE until all gel is covered.
3. Remove comb from the agarose gel carefully.
4. Then load samples into wells carefully in the correct order.
1st well: 3 μl of DNA ladder (Figure 1.1)
2nd well: Undigested plasmid DNA (All 6 μl)
3rd well: Digested plasmid DNA mix (All 6 μl)
5. Run samples at 100 V for 1 hour until bromophenol blue (purple dye) migrates at
least 2/3 of the distance.
6. Visualize and document under UV light.

18
 Figure 1.1 Example for a DNA ladder (2).

References
1. Sambrook, J., and David W. Russell, Molecular Cloning: A laboratory manual, third
edition, 3rd edition, volume 3. (Alkaline Lysis Solution Recipe and plasmid isolation
procedure are directly taken from this book)
2. http://www.fermentas.com/en/products/all/dna-electrophoresis/generuler-dna-
ladders/sm0313-generuler-1kb

19
Experiment-II: Amplification of TAT by PCR for TA-
cloning
Introduction
Polymerase Chain Reaction (PCR) and Gel Extraction
The Polymerase Chain Reaction (PCR) is a technique developed to amplify a specific
fragment of a nucleic acid strand based on the specific sequences at both ends of the fragment.
PCR reaction allows amplification of the specific fragment by over a million-fold, thus making
analysis possible using small amount of samples. PCR can be used for a wide range of purposes,
such as mutation analysis for diagnosis and DNA profiling for forensics. In this laboratory
session, PCR is going to be used to amplify the TAT gene that we aim to clone into TA vector
in the following laboratory session.
1. PCR Cycles
During PCR first the double-stranded template DNA need to be separated into single strands.
This step is called initial denaturation. This separation allows the primers to bind to the target
region. Achieving complete denaturation of the input DNA is important for ensuring efficient
amplification of the target sequence during the first amplification cycle. Typically, the initial
denaturation step is conducted at temperatures ranging from 94°C to 98°C for durations
spanning 1 to 3 minutes. The specific time and temperature parameters may vary based on
factors such as the nature of the template DNA and the salt concentrations of the buffer solution.
For instance, genomic DNA from mammalian sources may necessitate longer incubation
periods compared to plasmids and PCR products due to differences in DNA complexity and
size. Additionally, DNA sequences with high GC content, exceeding 65%, often require
extended incubation times or higher denaturation temperatures. Furthermore, addition of salt
leads to stabilization of DNA. Therefore, buffers containing high salt concentrations, which are
sometimes required by certain DNA polymerases, typically demand elevated denaturation
temperatures, such as 98°C, to effectively separate double-stranded DNA strands.
PCR is accomplished by repetitive cycles. In each cycle, the reaction goes through three
main steps: denaturation, annealing, and elongation. The cycles are repeated a certain number
of times, usually between 20 and 35, depending on the experiment. The fragment that is
produced at the end of each cycle serves as a template in the following cycle. Thus, the number
of the target DNA fragments increase by 2 in every cycle. So, in 20 cycles PCR reaction
produces more than 1 million (220) copies of the target DNA, while in 30 cycles the copy number
increases to 1 billion (230). The precise control of the reaction temperature and the incubation
times of each step in each cycle is achieved by the instrument called thermal cycler.
1. Denaturation: Every cycle starts with denaturation step. During this step, sample is
incubated at a high temperature (90 oC – 96 oC), which breaks the hydrogen bonds
between the bases. Thus, the double stranded DNA melts and gives rise two single
strands. Any newly synthesized strands are separated by starting each cycle with
denaturation. This ensures that the template strands are available for primer annealing
in the next cycle. Additives like glycerol, DMSO, formamide, and betaine can aid in the
separation of double-stranded DNA during denaturation in PCR. They enhance
specificity and facilitate efficient denaturation, reducing the necessity for longer
incubation periods or higher temperatures.
2. Annealing: During this phase, the temperature of the reaction is reduced to facilitate
the binding of primers to the target DNA. The annealing temperature is determined by
calculating the melting temperature (Tm) of the selected primers for PCR amplification.
20
 Typically, it is recommended to initiate with an annealing temperature that is 3–5°C
lower than the lowest Tm of the primers. The Tm represents the temperature at which
50% of the primer and its complementary sequence form a stable duplex. It's worth
noting that PCR additives can influence the Tm of the primer-template complex. For
instance, the presence of 10% DMSO can lower the annealing temperature by 5.5–
6.0°C. Primers, which are designed specific to the target sequences, have a specific
annealing temperature that depends on the primer size and sequence. Annealing
temperature can be in the range of 40 oC to 65 oC. Temperatures lower than the specific
annealing temperature may lead to unspecific binding of the primers to low-match
sequences. This results in the amplification of off-target DNA sequences and reduces
efficiency of the PCR reaction. Increasing annealing temperature increases specificity
of primer binding. However, temperatures over calculated optimum annealing
temperature will prevent DNA base pairing between matching sequences as well.
3. Elongation (Extension): After annealing of the primers, the reaction temperature is
raised to 72 oC, where thermostable DNA polymerase (e.g. Taq polymerase) starts
elongating the 3’ end of the primers by incorporating the dNTPs to synthesize
complementary DNA. The duration of the extension step in PCR is determined by the
speed at which DNA polymerase synthesizes DNA and the length of the target DNA.
For Taq DNA Polymerase, the standard extension time is approximately 1 minute per
kilobase (kb) of DNA. Longer DNA fragments require extended extension times to
ensure complete replication compared to shorter DNA sequences.
After completing the last cycle of PCR, the final extension step involves placing the PCR
mixture at the extension temperature, typically around 72°C, for a period lasting 5 to 15
minutes. The length of this step varies based on factors like the length and composition of the
target DNA fragment. It's crucial to optimize this duration to ensure complete polymerization
of the DNA and to obtain a satisfactory yield of the desired DNA product. Moreover, DNA
polymerases like Taq DNA polymerase, which possess terminal deoxynucleotide transferase
activity (TdT), can add extra nucleotides to the 3′ ends of the PCR products during this stage,
aiding in filling in incomplete ends.
At the end of the first cycle, the amount of the DNA fragments that contain target sequence
is doubled. However, the length of the newly synthesized fragments is longer than the target
sequence since there is no signal to end the synthesis (Figure 2.1 (a)). Newly synthesized DNA
fragments are used as templates during the second cycle, which produces the target sequence at
the intended length, the amplicon (Figure 2.1 (a)). During subsequent cycles, the amplicon
number increases at a higher rate due to the increased number of amplicons being used as
templates (Figure 2.1 (b)). After a certain number of PCR cycles (usually between 30 and 40
cycles, depending on the application), more than a billion copies of the target DNA are
produced.

21
(a)

Figure 2.1: First four cycles of PCR reaction. (a) Each step, denaturation, annealing, and
elongation are shown for the first two cycles. Red and blue lines indicate primers and newly
synthesized DNA, respectively. (Figure continues the next page)

22
(b)

Figure 2.1: First four cycles of PCR reaction. (b) End products at the end of third and fourth
cycles are shown. Yellow box represents the target region that is aimed to be amplified. Red
and blue lines indicate primers and newly synthesized DNA, respectively.
2. PCR Components
1. Template DNA: A PCR template can be obtained from various DNA sources such as
genomic DNA (gDNA), complementary DNA (cDNA), and plasmid DNA. Optimal input
amounts required for PCR amplification vary depending on the target region to be amplified
and the complexity of the template. For instance, a range of 0.1–1 ng of plasmid DNA is
typically adequate, whereas 5–50 ng of gDNA might be necessary for a 50 µL PCR reaction.
The optimal amount of template can also vary depending on the type of DNA polymerase
utilized; some DNA polymerases are engineered with increased sensitivity and therefore
may require less template. It is essential to optimize the DNA input as higher amounts

23
 increase the risk of nonspecific amplification, while lower amounts can decrease yields.
Integrity of the input DNA affects efficiency of the PCR reaction. In addition, template may
contain substances that interfere with PCR, acting as inhibitors. Thus, ensuring the purity
of the template is crucial.
2. Primers: PCR primers are synthetic DNA oligonucleotides typically consisting of 15–30
bases. Primers that bind to the antisense strand of the DNA are referred to as forward
primers. Primers that bind to the sense strand of the DNA are referred to as reverse primers.
These primers are designed to bind, based on sequence complementarity, to the sequences
flanking the region of interest within the template DNA. Ensuring the specificity of PCR
amplification requires careful primer design beyond mere sequence similarity. It is
important that primer sequences exhibit melting temperatures (Tm) falling within the 55°C
to 70°C range, with the Tm values of both primers being no more than 5°C apart. Moreover,
it is essential to design primers in a manner that precludes any complementarity between
them, particularly at their 3' ends, as such complementarity could lead to the formation of
primer-dimers. Additionally, it is critical to avoid self-complementarity, which may induce
self-priming and the formation of secondary structures. Moreover, it is recommended that
the GC content of the primer falls within the 40–60% range, with an even distribution of C
and G bases throughout to prevent mispriming. It is important to limit the presence of no
more than three G or C bases at the 3′-ends of the primers to reduce nonspecific priming.
However, having one C or G nucleotide at the 3′ end of a primer can facilitate favorable
primer anchoring and extension because they form a more stable bond compared to A and
T (3 hydrogen bonds between C and G vs 2 hydrogen bonds between A and T). To simplify
the process, several online tools are available for bioinformatic primer design and selection
based on specific parameters (Primer Blast: https://www.ncbi.nlm.nih.gov/tools/primer-
blast/). Additional sequences like restriction sites can be added as extensions to the 5′ ends
during the design of cloning primers. It's generally advisable to use primer concentrations
ranging from 0.3–1 μM. Elevated primer concentrations may lead to mispriming and
nonspecific amplification, while low concentrations could yield insufficient amplification
or no amplification for the desired target.
3. DNA polymerase: A thermostable DNA polymerase, such as Taq DNA polymerase, is
required for PCR reaction to elongate the primers that are bound to the target DNA in a
sequence specific manner. Thermostable DNA polymerase elongates primers by adding
nucleotides complementary to the template sequence during elongation step.
4. dNTPs: dNTPs, the deoxynucleoside triphosphate, are the four types nucleotides that are
used by DNA polymerase to synthesize new DNA strand. Each dNPT is added at the final
concentration of 200 µM to the reaction mix. However, depending on the application a
different concentration (between 50 to 500 µM) could be used.
5. Buffer, Magnesium, and Ammonium Sulfate: PCR is carried out within a buffer that
creates an optimal chemical environment for DNA polymerase activity. Typically, the
buffer maintains a pH range of 8.0 to 9.5 and is frequently stabilized using Tris-HCl.
Magnesium chloride and ammonium sulfate are some of the common components of Taq
DNA polymerase buffers. Magnesium ions act as cofactors for DNA polymerase by
facilitating the incorporation of dNTPs during polymerization. Within the enzyme’s active
site, magnesium ions catalyze the formation of phosphodiester bonds between the 3’-OH of
a primer and the phosphate group of a dNTP. Moreover, Mg2+ assists in the formation of
complexes between primers and DNA templates by stabilizing the negative charges present
on their phosphate backbones. Final concentration of MgCl2 in a reaction varies depending
on the PCR conditions. Increase MgCl2 concentrations are required for templates
containing PCR inhibitors (e.g. EDTA) since they also bind to Mg2+ ions and reduce their

24
 availability. Very high or very low levels of MgCl2 can have adverse effects on PCR.
Elevated levels of magnesium chloride in PCR facilitate non-specific primer binding and
increase the likelihood of primer dimer formation. Conversely, reduced magnesium
chloride concentrations hinder primer-base pairing with the DNA template, causing weak
amplification or complete reaction failure. (NH4)2SO4 increases specificity of PCR. The
presence of the ammonium ion (NH4+) increases specificity by destabilizing weak
hydrogen bonds, particularly those occurring between mismatched primer-template base
pairs.
In this laboratory section, you will amplify TAT gene using pCV-1 plasmid with the insert,
which you isolated in the last lab session, as a template. Then, you will run the PCR product on
agarose gel and perform agarose gel extraction to prepare it for TA cloning that you will do in
the next lab session.

Materials and Methods

Primer Sequences
TAT-Bam5: 5’ – GGA TCC ACC ATG GAG CCA GTA GAT CC -3’
TAT-Eco3: 5’ – GAA TTC TAT TCC TTC GGG CC-3’

The underlined sequences are restriction sites for BamHI and EcoRI. “ATG” and “ATT” in
squares are “start” and “stop” codons for TAT gene, respectively.
For PCR and Agarose Gel
o Template DNA
o 10X Buffer with with (NH4)2SO4 and 20 mM MgCl2 for Taq DNA polymerase
o Taq DNA polymerase
o Magnesium chloride
o dNTP mix
o Forward and Reverse primers
o Nuclease free dH2O
o Template pCV-1 plasmid containing TAT gene
o Agarose
o TBE (Tris- Boric Acid- EDTA) solution
o GeneRuler 1 kb DNA Ladder

For extraction from gel

o GeneJET Gel Extraction Kit

Safety
1. You will be doing gel extraction in this lab session, which includes cutting the DNA
band under UV light. UV exposure is hazardous. The eye is the most vulnerable organ
and short term UV exposure results in painful eye. Appropriate shield and eye protection
should be used. UV exposure also can cause skin burns. While handling something under
UV light, all skin must be covered and there should not be any gap in the protective
clothing.

25
 2. While handling any experimental staff (pipette, Erlenmeyer, eppendorf, etc) always be
aware of wearing gloves.
3. Ethidium bromide is a mutagen and always wear gloves while handling all staffs possibly
contaminated with ethidium bromide.
4. Never touch and use any staff in the lab without permission and information from your
lab assistants.
5. Agarose boiled in microwave can easily boil up to overflow the flask. Swirl gently with
caution.

Protocol
PCR Reaction for TAT gene
1. Previously isolated pCV-1 plasmid including TAT gene will be used as a template. The
volume of template used changes depending on its concentration.
2. 2 reactions per group will be set up. A master mix that includes all the reagents (Table
2.1) will be prepared for 3 reactions to avoid shortcoming due to pipetting errors. All
reagents except Taq polymerase should be thawed on ice, gently vortexed and briefly
centrifuged. Taq polymerase should be added the last and must be taken out of -20 oC
shortly before adding to the master mix. Briefly vortex and then spin down the master
mix.
3. Distribute one reaction volume (25 µl) to PCR tubes and place them into the PCR
machine.
PCR reaction conditions are as following:

Initial Denaturation: 95 oC for 3 min

Denaturation : 95 oC for 30 sec

Annealing : 53 oC for 30 sec 30 cycle
Extention : 72 oC for 30 sec

Final Extention : 72 oC for 5 min

Table 2.1 PCR reaction components and volume

Component Final Volume
Concentrations
10X Taq Buffer with (NH4)2SO4 1 X
and 20 mM MgCl2
dNTP mix, 2mM each 0.2 mM of each
25 mM MgCl2 0.5 mM
Forward primer (10 µM) 0.5 µM

Reverse primer (10 µM) 0.5 µM

Template 1 ng
Taq DNA Polymerase (5 U/µl) 1.25 U
Nuclease-free dH2O to 25 µl
TOTAL - 25 µl

26
Agarose Gel Electrophoresis
1. 1% agarose gel is prepared as explained in Experiment-I.
2. GeneRuler 1 kb DNA Ladder will be loaded into the first well. Each PCR product, 25
µl, is mixed with ............. µl of 6x loading dye (final concentration of dye will be 1x)
and loaded into the wells of agarose gel.
3. Run agarose gel at 100 Volt for 60 minutes.

Figure 2.2: GeneRuler 1 kb DNA Ladder

Gel Extraction
GeneJET Gel Extraction Kit will be used for this purpose. (Protocol is directly taken from
the manual of the kit)
1. Excise gel slice containing the fragment carefully to minimize the gel volume. Determine
the weight of the gel slice and transfer it to a clean tube.
2. For each 100 mg of agarose gel add 100 μl Binding buffer. Incubate sample at 50-60 °C
until the gel slices are dissolved (10 min). Vortex the sample briefly every 2-3 min until
the gel slices is completely dissolved and before loading on the column!

27
 3. Check the color of the solution. A yellow color indicates an optimal pH for DNA binding.
If the color of the solution is orange or violet, add 10 µL of 3 M sodium acetate, pH 5.2
solution and mix. The color of the mix will become yellow.
4. Optional: use this step only when DNA fragment is ≤500 bp. If the DNA fragment is
≤500 bp, add 1 gel volume of 100% isopropanol to the solubilized gel solution (e.g. 100
µL of isopropanol should be added to 100 mg gel slice solubilized in 100 µL of Binding
Buffer). Mix thoroughly.
5. To bind DNA, place a GeneJET purification column into a 2 ml collecting tube and load
up to 800 µL of the solubilized gel solution for each time. Centrifuge for 1 min at 12,000
x g. Discard flow-through and place the GeneJET purification column back into the
collecting tube.
6. To wash silica membrane, add 700 μl Wash Buffer. Centrifuge for 1 min at 11,000 x g.
Discard flow-through and place the GeneJET purification column back into the
collecting tube.
7. To dry silica membrane, centrifuge for 1 min at 12,000 x g to remove Wash Buffer
quantitatively. Make sure the spin column doesn’t come in contact with the flow-through
while removing it from the centrifuge and the collecting tube.
8. As a final to elute DNA Place the GeneJET purification column into a clean 1.5 ml
microcentrifuge tube. Add 50 μl elution buffer and incubate at room temperature for 1
min to increase the yield of eluted DNA. Centrifuge for 1 min at 12,000 x g.
9. Discard the GeneJET purification column and store the purified DNA at -20 °C.

References
1. JF Sambrook (2001) Molecular cloning: A laboratory manual Cold Spring Harbor
Laboratory
2. web.chemistry.gatech.edu
3. Thermofisher.com

28
Experiment-III: TA Cloning of TAT

Introduction
Joining of DNA molecules
DNA ligation is a critical step in many molecular biology workflows. The sealing of
the single stranded nicks between adjacent residues of a double stranded DNA are facilitated,
catalyzed by DNA ligases. Commercially used DNA ligases are encoded by E. coli and phage
T4. In living organisms, DNA ligases are also critical with their roles in DNA repair and
replication. The DNA ligase complex binds to the nick, which expose free 5’ end 3’ ends with
phosphate and OH groups respectively. Then ligase complex joins the two ends by making a
phosphodiester bond. The logic behind the ligation of the vector and gene of interest in the
laboratory is the same. But this time, same reactions occur in the tubes, which contains ligase
enzyme, vector, gene to be ligated, buffer to adjust the pH for optimum working condition of
the enzyme.
Cloning of sticky ended DNA fragments is more efficient than cloning of blunt ended DNA
fragments, but components of the ligation reaction are the same. For sticky ended ligation, there
is no oriantation problem. But in blunt ended ligation, joining of two ends is random, because
enzyme only joins 5’ phosphate and 3’ OH groups, without any specific direction. But in sticky
ended ligation, sticky ends complement each other at the right directions. Also H bonds occur
between these complementary ends between vector and the gene to be cloned and this also
increases the efficiency of the reaction. Therefore, by using sticky ended ligation rather than
the blunt ended one, we can decrease the incubation time, and DNA amounts in the ligation.
TA Cloning
In this laboratory section, we will perform TA cloning by joining TAT gene and pTZ57R/T
vector (Figure 3.1). TA cloning is one of the most effective and the simplest methods for the
cloning of PCR products. TA cloning takes advantages of the terminal transferase activity of
thermophilic DNA polymerases. Thermus aquaticus (Taq) polymerase used in PCR is
thermophilic DNA polymerase and has non-template dependent activity which leads to
preferentially adding of a single adenosine to the 3'-ends of a double stranded DNA molecule.
Therefore, the molecules amplified with PCR mostly have a single 3'A overhangs.
Another important component used in TA cloning is linearized “T-vector” and acquired its
name from its possessed 3’T overhangs on both ends. T-vector allows high-efficient cloning of
PCR products that is mediated by the complementarity between 3’A overhangs of PCR products
and 3’T overhangs of the T-vector. The TA cloning can be adapted to different DNA fragment
species easily as the same T-vector can be used to clone both blunt and sticky ended DNA
fragments. TA cloning is especially useful with complementation between vector and DNA
fragment when DNA fragments that are lack of compatible restriction sites are subcloned from
one vector to another which is not accomplishable. In consequence of TA cloning, PCR product
can be inserted in both directions. Directional cloning can be achieved by appropriate hemi-
phosphorylation of both the T-vectors and the inserts.
All in all, any DNA fragment can be cloned without compromising the cloning efficiency
with a single T-vector at hand. TA cloning method is both convenient and labor-saving.

29
 a)

b)

Figure 3.1. TA cloning. a) Schematic explanation of TA cloning protocol. b) Map of pTZ57R/T

cloning vector (http://tools.thermofisher.com/)
Transformation
After ligation reaction of TAT gene and T-vector, plasmid DNA will be introduced to E. coli
cells. And transformation is the procedure to introduce a foreign DNA molecule (in this case
ligated TAT-T vector) to an organism such as bacteria, yeast or plant cells. There are two
important transformation techniques:
1) Electroporation
2) Heat Shock Method
1) Electroporation: Electroporation is based on fast and simple process for introducing
cloned genes into a wide range of microbial, plant and animal cells, including E. coli. It is a
simple and fast method that enables transformation of a large number of cells in a short time
when optimal electroporation conditions are determined. High-voltage electric pulses disturb
the lipid bilayer leading to formation of temporary pores in the cell membranes. Subsequently,
cells take up charged molecules as exogenous DNA from suspending solution. One of the main

30
advantages of the electroporation is based on its applicability for transient and stable
transformation of all cell types. However, high voltage pulses can lead to substantial cell death
and partial membrane repairing which requires the usage of greater quantities of the cells.
2) Heat Shock Method: In this method, cells known as competent cells are stored in -800C
in a solution containing calcium ions. After adding of plasmid DNA to the competent cells,
positive charges of Ca2+ ions shields negative charge of DNA phosphates and thus, DNA
molecules can attach to the surfaces of the cells. Incubation of cells on ice also slows fluid cell
membrane. Lastly, heat shock increases permeability of membranes. So, foreign DNA molecule
enters the cells more efficiently. After heat shock, incubation of cells with SOC medium before
plating on the antibiotic containing medium is important. During this incubation, phenotypic
properties conferred by the plasmid such as antibiotic resistance genes are expressed. Thus,
when the cells are plated on antibiotic containing plate, the ones that contain the plasmid and
expressed the resistance gene survive and form colonies.
Blue-white screening (X-gal screening)
Ligation solution that is added to the competent cells also contains undesired DNA fragments
in addition to TAT/T-vector and cells can grow on selective medium as shown in figure.
Therefore, it is important to select positive clones on the plate.
a)

b)

Figure 3.2. Blue-white screening. Schematic representation of a) reporter plasmid (pTZ57R/T

for this experiment) and b) screening protocol. (https://www.sigmaaldrich.com)

31
What is blue-white screening and α-complementation?
Genetic engineering of “lac” operon in the Escherichia coli laboratory strain serve as a
recombination marker with subunit complementation achieved with cloning vector. Normally
the presence of the lactose in surrounding environment triggers the production of the β-
galactosidase enzyme which metabolizes the lactose in Escherichia coli. By genetically
engineering of lac operon some residues in the lacZ gene is mutated and it is unable to form
functional β-galactosidase enzyme. The plasmid vector then modified in such a way and due to
the complementation of the one subunit of the LacZ protein encoded by plasmid vector β-
galactosidase enzyme activity is rescued.
The vector that used in cloning is manipulated as α subunit of LacZ protein with an internal
multiple cloning site (MCS) is encoded by the vector (e.g. pBluescript) while the remaining Ω
subunit is encoded by the chromosome of the host strain and in the presence of both subunit
functional β-galactosidase enzyme forms. A multiple cloning site located within the lacZ
sequence in the vector can be cleaved by different restriction enzymes to insert foreign DNA
and if foreign DNA is inserted into the "lacZ"α gene, the α-complementation cannot be
expressed, therefore, a functional β-galactosidase enzyme is not produced.
The reagent used to detect whether there is functional β-galactosidase enzyme is X-gal. X-
gal is a colorless modified galactose that forms an insoluble product which is bright blue when
metabolized by β-galactosidase. If "lacZ"α gene is not disrupted and functional β-galactosidase
is produced, colorless X-gal is hydrolyzed, and blue pigments are produced which leads to
appearing of blue colonies. These blue colonies represent non-recombinant cells. If foreign
DNA is inserted, cells lose their ability to hydrolyze X-gal substrate and appeared in white
color. Thus, X-gal functions as an indicator, which allows appearance of colonies with no
recombination as blue and with recombination (where an insert is placed into the MCS) as
white.
Isopropyl β-D-1-thiogalactopyranoside (IPTG), which is a non-metabolizable analog of
galactose, works as an inducer of the Lac operon (Figure 3.2). Although many laboratory strains
do not require IPTG, it can be used in some strains to enhance the phenotype by increasing the
activity of the operon. Since IPTG cannot be metabolized, it is only an inducer and not a
substrate.

Materials and Methods

Ampicillin Stock Solution (50 mg/ml)
1) Dissolve 2.5 g of ampicillin sodium salt in 50 ml of deionized water.
2) Filter-sterilize and store in aliquots at -20°C.
X-Gal Stock Solution (20 mg/ml)
1) Dissolve 200 mg X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) in 10 ml
N,N-dimethylformamide.
2) Store at -20°C in the dark.
IPTG stock solution (100 mM)
1) Dissolve 1.2 g IPTG (isopropyl- β -D-thiogalactopyranoside) in 50 ml deionized water.
2) Filter-sterilize, aliquote and store at 4°C.

32
LB-ampicillin X-Gal/IPTG Plates
1) Prepare LB-agar medium (1 liter), weigh out:

 10 g Tryptone

 5 g Yeast extract

 10 g NaCl
2) Dissolve in 800 ml of water, adjust pH to 7.0 with NaOH and adjust the volume with
water to 1000 ml.
3) Add 15 g of agar and autoclave to sterilize.
4) Before pouring the plates, allow the medium to cool to 55°C. Then, add 1 ml of ampicillin
stock solution (50 mg/ml) to a final concentration of 50 µg/ml.
5) Mix gently and pour the plates. Allow the LB-ampicillin agar medium to solidify.
6) Add 40 µl of X-Gal solution (20 mg/ml) and 40 µl of IPTG 100 mM, spread evenly with
a sterile spatula.

Safety
1. While handling any experimental staff (pipette, Erlenmeyer, eppendorf, etc) always be
aware of wearing gloves.
2. Ethidium bromide is a mutagen and always wear gloves while handling all staffs possibly
contaminated with ethidium bromide.
3. Never touch and use any staff in the lab without permission and information from your
lab assistants.
4. Agarose boiled in microwave can easily boil up to overflow the flask. Swirl gently with
caution.

Protocol
TA Cloning / Ligation Reaction
IMPORTANT NOTES

 DNA length of TAT gene: around 270 bp

 pTZ57R/T vector length: 2886 bp

 The optimal insert/vector ratio is 3:1.

 Required vector DNA mass for efficient ligation with 0.165 μg of the pTZ57R/T vector
according to manufacturers.

 Calculate required mass insert using formula below.

33
 1) Mix the following in a PCR tube:

 3 µl of pTZ57R/T vector (0.165 µg)

 3 µl of 10X ligation buffer (working 1X)

 X µl of insert DNA (46 ng is needed according to 3:1 molar ratio)

 1 µl of T4 DNA ligase
2) Add nuclease-free dH2O into the tube to make final reaction volume 30 µl.

3) The ligation mixture is incubated for 1 h at 22°C.

Transformation
1) E. coli cells are incubated at ice for 2 minutes.
2) 5 µl of ligation mixture is added onto cells.
3) This cell-ligation mixture is incubated on ice for 25 minutes.
4) The cells are are taken to waterbath at 420C for 50 seconds.
5) The cells are immediately taken onto ice for 2 minutes.
6) 250 µl of LB medium is added onto the cells.
7) The cells are shaken at 37oC and 200 rpm for 1 hour.
8) Then, the cells are spread on LB-Amp/IPTG/X-GAL plates as 100 µl.
9) Next day, white colonies are selected by colony PCR reaction for further confirmation of
cells whether they really contain TAT gene or not, in another words, colony PCR is done in
order to analyze recombinant clones.

34
Colony PCR
1) Mix the following in a PCR tube.

Component Volume
10X Taq Buffer 2.5 ul
dNTP mix, 2mM each 1 ul
25 mM MgCl2 2.5 ul
Forward primer 2 ul
Reverse primer 2 ul
Taq Polymerase 0.5 ul
Nuclease-free dH2O 14.5 ul
TOTAL 25 ul

2) An individual white colony is picked by pipette tip, and resuspended in the mixture.
3) PCR is performed at the following conditions
Colony PCR conditions
Initial Denaturation: 94oC for 10 min
Denaturation : 94oC for 30 sec
Annealing : 53oC for 30 sec 30 cycle
Extention : 72oC for 45 sec
Final Extention : 72oC for 5 min

4) PCR samples are analyzed on an agarose gel 1% concentration for the presence of the
PCR product of the expected length.

References
1) Curr. Issues Mol. Biol. (2000) 2(1): 1-7.
2) Priciples of Gene Manipulation, 6th Edition; Primrose, Twyman and Old.
3) dic.academic.ru
4) J. Mol. Biol. (1983) 166(4):557-580.
5) http://tools.thermofisher.com/content/sfs/manuals/MAN0012706_InsTAclone_PCR_Cl
oning_UG.pdf
6) https://www.sigmaaldrich.com/technical-documents/articles/biology/blue-white-
screening.html

35
Experiment-IV: Subcloning of TAT from TA Vector to
PGEX2 Expression Vector
Introduction
Production level of each protein varies in different cell types and conditions. Regulation of
transcription and translation machinery determines their amount. For an efficient yield, cloning
techniques take advantage of using a strongly regulatable promoter in delivery systems. Under
the control of this promoter, concentration of protein of interest can be adjusted. Protein is
present at high levels in case of induction while its amount is diminished after switch off.
An expression vector is used to introduce and express a specific gene into a target cell.
It provides the insertion, storage and expression of the exogenous DNA, by this way we could
obtain large amounts of proteins. The gene carried by vector provides information to build the
protein with host cell’s transcription and translation machinery. To enhance transcription step,
those engineered vectors have well-designed regulatory elements, specifically enhancers. In
order to obtain high amounts of protein, the gene of interest should be cloned downstream of a
strong promoter with appropriate enhancers. These promoters usually control genes whose
translation products are required in large amounts by the cell. Also, promoter should be
regulated by the cell itself. There are two types of gene regulation, induction and repression.
An inducible gene is one whose transcription is switched on by addition of a chemical to the
growth medium. On the other hand, presence of a compound can trigger shutdown of gene
expression in the case of repression. Although most of the regulatory sequences are located
around promoter region, enhancer and silencer sections, which are the binding sites for
activators and repressor, respectively, can be found along the gene. Moreover, an expression
vector should contain a ribosomal binding sequence, a protease cleavage site and a terminator
sequence which will be recognized by the host organism.
pGEX-2T is widely used as an expression vector (Figure 4.1) that provides a strong
promoter which is called as a Tac promoter. Tac promoter is a hybrid promoter, which is
composed of -35 region of tryptophan promoter and -10 region of the lacUV5
promoter/operator of E. coli. This expression vector consists of mainly Tac promoter, lacIq
repressor, GST (Glutathione S-transferase) coding sequence to produce a fusion protein,
protease cleavage recognition sites.
Tac promoter is about ten fold more efficient in transcription initiation compared to the
wild type promoter. Expression of this vector can be upregulated by the lactose analog isopropyl
b-D thiogalactoside (IPTG), in other words, Tac promoter can be induced by IPTG. There are
thirteen types of pGEX-2T vector and all vectors are also engineered with an internal lacIq
gene.
The lacIq gene product is a repressor protein that binds to the operator region of the tac
promoter, preventing its expression. Promoter-up mutation of the LacI gene increases
production of LacI by ten fold resulting in strong repression of tac promoter until induction by
IPTG, thus maintaining tight control over expression of the insert.
High-level expression proteins which are expressed by pGEX-2T, are expressed as
fusion proteins with the 26 kDa Glutathione S-transferase (GST). The GST gene contains an
ATG (start codon) and ribosome-binding site, and is under control of the tac promoter and
translation terminator is provided in each reading frame.

36
 Also each pGEX-2T vectors have a range of protease cleavage recognition sites allow
for removal of the GST carrier protein from the fusion by enzymatic cleavage with
PreScission™ Protease, thrombin and factor Xa. pGEX-2T expression vector confers
resistance to 100 µg/ml of ampicilline and ampicilline resistance gene is used as a selectable
marker because it is not possible to determine transformed colonies by blue-white screening
method.
pGEX-2T expression vector is 4986 bp in length and the maximum insert size based
purely on cloning limitations would be that the vector and insert be no more than a total of 10-
11 kb. Insert including an open reading frame must have matching termini sites with destination
vector. Formation of noncomplementary ends at both vector and insert as a result of double
digestion using two distinct enzymes, prevents orientation problems during cloning. Thus,
transfer of insert into vector occurs in the right direction. Additionally, self ligation possibility
is eliminated. This process is named as directional cloning.
Majority of E.coli strains might be utilized as the host cell. Modifications in those strains
may improve expression level. Besides, strains lacking cytoplasmic proteases such as Lon,
OmpT, DegP, HtpR should be preferred to maintain high concentration of the protein. For
instance, E. coli BL21 is an OmpT-deficient strain so it is widely used for expression of GST
fusion proteins. The strains positive for RecA is not preferred due to the increased rate of
positional changes or deletions in plasmid sequences.
GST tag is advantageous to isolate the fusion proteins expressed in bacteria. Depending on
enzyme-substrate interaction, affinity chromatography is carried out with glutathione coupled
beads to bind to and isolate GST fusion proteins. Detection of GST-tagged fusion proteins can
be achieved through microplates coated with Glutathione or anti-GST, ELISA or Western
blotting using anti-GST antibodies, or other enzymatic assays such as CDNB, which is a
substrate for GST enzyme.

Figure 4.1 pGEX-2T vector map (https://www.sigmaaldrich.com)

Double Digestion is the process of cutting DNA by two restriction endonucleases at once.
Two enzymes can be added to the same reaction if the working conditions such as pH or

37
temperature are compatible. Otherwise, reaction of each restriction enzyme must be performed
sequentially with their own appropriate conditions. In double digestion the reaction conditions
have a vital importance. Since we are working with enzymes, choosing the optimum buffer
conditions determine the activities of enzymes which in parallel determines the efficiency of
our experiment.
Digestion reaction can reach to 100% activity using recommended buffer. Optimal buffer
also facilitates recognition fidelity of the enzyme, thereby limits possibility of star activity.
Under unfavorable conditions, enzymes tend to recognize the sequences resembling their
recognition site. This change in specificity is referred to as star activity. Other situations that
causes star activity other that non optimal buffer are also
-high level of glycerol concentration,
-high concentration of enzyme/µg of DNA ratio, ,
- prolonged incubation time,
-presence of organic solvents and
-substitution of Mg +2 with other divalent cations.
Each enzyme is supplied with its optimal buffer to ensure 100% activity. It is important to
check the activity chart for rates the percentage activity of each restriction endonuclease in
different buffer systems before set up restriction reaction (Figure 4.2). Each supplier provides
charts for its own products.

Figure 4.2 Reaction conditions of BamHI and EcoRI enzymes from Fermentas.
(https://www.thermofisher.com)
Tango buffer is a special buffer that is especially used in double digestion reactions. This
buffer provides the optimum medium for the activity of BamHI and EcoRI restriction enzymes
preventing the star activity.

Safety
1. While handling any experimental staff (pipette, Erlenmeyer, eppendorf, etc) always be
aware of wearing gloves.
2. Ethidium bromide is a mutagen and always wear gloves while handling all staffs possibly
contaminated with ethidium bromide.

38
 3. Never touch and use any staff in the lab without permission and information from your
lab assistants.
4. Agarose boiled in microwave can easily boil up to overflow the flask. Swirl gently with
caution.

Materials and Methods

Tango Buffer (1X buffer composition)
33 mM Tris-acetate (pH 7.9 at 37°C),
10 mM magnesium acetate,
66 mM potassium acetate,
0.1 mg/ml BSA.
SOC Medium: This is a medium especially used for the recovery of E.coli competent cell
transformations.
- 0.5 % Yeast extract
- 2 % Tryptone
- 10 mM KCl
- 10 mM MgCl2
- 10 mM MgSO4
- 20 mM Glucose
Protocol
Growth of cells:
Before the lab session, positive colonies of E. Coli DH5-α strain which were transformed
with pTZ57R/T that contains ‘Tat Gene’ and selected by Blue-White colony screening method
previous week were inoculated to LB ampicillin plates and incubated overnight at 37°C. Then
colonies were inoculated into 3 ml of liquid medium which was containing ampicillin from
plates and incubated overnight at 37°C with vigorous shaking. Cultured cells were centrifuged
at maximum speed for 30 seconds and supernatant is removed. Lastly pellets were kept at -
20°C until plasmid isolation.
In this laboratory ‘’NucleoSpin® Plasmid Kits’’ will be used during plasmid isolation step.
According to kit’s manuel, following isolation protocol will be applied. (Protocol is directly
taken from the supplier’s manuel.)
Procedure for isolation of cloning vector from Transformed E. Coli DH5-α strain
GenElute™ Plasmid Miniprep (Sigma-Aldrich) will be used for this purpose. (Protocol is
directly taken from the manual of the kit)
Collect the pellet of 1-5 mL of an overnight recombinant E. coli culture by centrifugation.
Transfer the appropriate volume of the recombinant E. coli culture to a microcentrifuge tube
and pellet cells at ;12,000 x g for 1 minute. Discard the supernatant.
1) Resuspend cells
Completely resuspend the bacterial pellet with 200 μl of the Resuspension Solution. Vortex
or pipette up and down to thoroughly resuspend the cells until homogeneous. Incomplete
resuspension will result in poor recovery.
2) Lyse cells Lyse the resuspended cells by adding 200 μl of the Lysis Solution. Immediately
mix the contents by gentle inversion (6–8 times) until the mixture becomes clear and viscous.

39
Do not vortex. Harsh mixing will shear genomic DNA, resulting in chromosomal DNA
contamination in the final recovered plasmid DNA. Do not allow the lysis reaction to exceed
5 minutes.
Prolonged alkaline lysis may permanently denature supercoiled plasmid DNA that may
render it unsuitable for most downstream applications.
3) Neutralize Precipitate the cell debris by adding 350 μl of the Neutralization/Binding
Solution. Gently invert the tube 4–6 times. Pellet the cell debris by centrifuging at ;12,000 x g
or maximum speed for 10 minutes. Cell debris, proteins, lipids, SDS, and chromosomal DNA
should fall out of solution as a cloudy, viscous precipitate. If the supernatant contains a large
amount of floating particulates after centrifugation, recentrifuge the supernatant before
proceeding to step 6.
4) Prepare Column Insert a GenElute Miniprep Binding Column into a provided
microcentrifuge tube, if not already assembled. Add 500 μl of the Column Preparation Solution
to each miniprep column and centrifuge at 12,000 x g for 30 seconds to 1 minute. Discard the
flow-through liquid.
5) Load cleared lysate Transfer the cleared lysate from step 3 to the column prepared in
step 4 and centrifuge at; 12,000 x g for 30 seconds to 1 minute. Discard the flow-through liquid.
6) Optional wash (use only with EndA+ strains)
Add 500 μl of the Optional Wash Solution to the column. Centrifuge at ;12,000 x g for 30
seconds to 1 minute. Discard the flow-through liquid.
7) Wash column Add 750 μl of the diluted Wash Solution to the column. Centrifuge at
;12,000 x g for 30 seconds to 1 minute. The column wash step removes residual salt and other
contaminants introduced during the column load. Discard the flow-through liquid and
centrifuge again at maximum speed for 1 to 2 minutes without any additional Wash Solution to
remove excess ethanol.
8) Elute DNA Transfer the column to a fresh collection tube. Add 100 μl of Elution Solution
or molecular biology reagent water to the column. For DNA sequencing and other enzymatic
applications, use water or 5 mM Tris-HCl, pH 8.0, as an eluant. Centrifuge at;12,000 x g for 1
minute. The DNA is now present in the eluate and is ready for immediate use or storage at –20
°C.
9) Determine precise yields and integrity of isolated plasmid DNA by UV spectrophotometry
or Nanodrop.

Procedure for Double Digestion of Cloning Vector ‘pTZ57R/T

1) 2 µl of BamHI Restriction Enzyme
2) 1 µl of EcoRI Restriction Enzyme
3) 5 µl of Tango (2x) Buffer
4) Nearly 1000 ng of plasmid DNA
5) Water up to 25 µl

40
 6) Incubation at 37 °C for 1h
7) Load Restriction Reaction Samples to 1% concentration agarose gel

Figure 4.3 Expected bands which should be observed on Agarose gel. Bands which are above
on agarose gel nearly 3 kb and bands which are below on agarose gel nearly 250bp according
to marker (Fermentas - geneRuler™ 1kb DNA Ladder)
Procedure For Purification of Tat Gene From Agarose Gel
In this step “GenElute™ Gel Extraction Kit’’ (Sigma) will be used. According to kit’s
manuel, following isolation protocol will be applied. (Protocol is directly taken from the
supplier’s manuel)
All centrifugations (spins) are performed at 12,000 to 16,000 x g.
1) Excise band. Excise the DNA fragment of interest from the agarose gel with a clean,
sharp
scalpel or razor blade. Trim away excess gel to minimize the amount of agarose.
2) Weigh gel. Weigh the gel slice in a tared colorless tube.
3) Solubilize gel. Add 3 gel volumes of the Gel Solubilization Solution to the gel slice. In
other
words, for every 100 mg of agarose gel, add 300 µl of Gel Solubilization Solution. Incubate
the gel mixture at 50-60 °C for 10 minutes, or until the gel slice is completely dissolved. Vortex
briefly every 2-3 minutes during incubation to help dissolve the gel.
4) Prepare binding column. Preparation of the binding column can be completed while the
agarose is being solubilized in step 3. Place the GenElute Binding Column G into one of the
provided 2 ml collection tubes. Add 500 µl of the Column Preparation Solution to each binding
column. Centrifuge for 1 minute. Discard flowthrough liquid.
5) Check the color of the mixture. Once the gel slice is completely dissolved (step 3) make
sure the color of the mixture is yellow (similar to fresh Gel Solubilization Solution with no gel
slice) prior to proceeding to the following step. If the color of the mixture is red, add 10 µl of
the 3 M Sodium Acetate Buffer, pH 5.2, and mix. The color should now be yellow. If not, add
the 3 M Sodium Acetate Buffer, pH 5.2, in 10 µl increments until the mixture is yellow.
6) Add isopropanol. Add 1 gel volume of 100% isopropanol and mix until homogenous.
For a gel with an agarose concentration greater than 2%, use 2 gel volumes of 100%
isopropanol.

41
 7) Bind DNA. Load the solubilized gel solution mixture from step 6 into the binding column
that is assembled in a 2 ml collection tube. It is normal to see an occasional color change from
yellow to red once the sample is applied to the binding column. If the volume of the gel mixture
is >700 µl, load the sample onto the column in 700 µl portions. Centrifuge for 1 minute after
loading the column each time. Discard the flow-through liquid.
8) Wash column. Add 700 µl of Wash Solution (diluted from Wash Solution Concentrate
G as described under Preparation Instructions) to the binding column. Centrifuge for 1 minute.
Remove the binding column from the collection tube and discard the flow-through liquid. Place
the binding column back into the collection tube and centrifuge again for 1 minute without any
additional wash solution to remove excess ethanol. Residual Wash Solution will not be
completely removed unless the flow-through is discarded before the final centrifugation.
9) Elute DNA. Transfer the binding column to a fresh collection tube. Add 50 µl of Elution
Solution to the center of the membrane and incubate for 1 minute. Centrifuge for 1 minute. For
efficient recovery of intact plasmid DNA, preheat the elution solution to 65 °C prior to adding
it to the membrane. Eluting at 65 °C improves plasmid DNA recoveries by 2 to 3-fold. Yields
of large linear DNA fragments (>3 Kb) can also be increased by up to 20% by preheating the
elution solution to 65 °C.
10) Determine yields of purification procedure by UV spectrophotometry or Nanodrop.
Procedure for Double Digestion of Expression Vector ‘’pGEX-2T’’
•2 µl of BamHI Restriction Enzyme
•1 µl of EcoRI Restriction Enzyme
•5 µl of Tango (2x) Buffer
•Nearly 1000 ng of plasmid DNA
•Water up to 25 µl
•Incubation at 37 °C for 1,5 h
•Load Restriction Reaction Samples to 1% concentration agarose gel

Figure 4.4 Expected bands which should be observed on agarose gel. Bands are nearly 5 kb
according to marker (Fermentas - geneRuler™ 1kb DNA Ladder)

42
Procedure For Purification of Expression Vector ‘’pGEX-2T’’
The Plasmid MiniPrep kit from Sigma will be used and the experiment will be done
according to the protochol. Determine yields by UV spectrophotometry or Nanodrop.
Ligation and transformation of Tat gene and pGEX-2T plasmid
After purification of pGEX2T plasmid and Tat gene, ligation reaction is performed.
1) 2 µL T4 DNA ligase buffer
2) pGEX-2T plasmid (calculate the required amount from Nanodrop result of plasmid)
3) Tat gene (calculate the required amount from Nanodrop result of Tat gene)

The ratio for pGEX-2T/Tat gene should be 1:3 to increase the chance for ligation!!!!
4) 1 µL T4 DNA ligase
5) Water up to 20 µL
After mixing the above components incubate the tubes at 14 oC for 1 hour.After incubation
period add 5 µL from 2 ligation mixture to 2 different 100 µL BL21 competent cell containing
tubes. Then incubate the tubes for 30 minutes on ice. Later place the tubes at 42 oC waterbath
for 45 seconds. After that put them on ice for 2 minutes. Finally, add 200 µL of SOC medium
to each tube and then incubate them at 37 oC for 1 hour with a speed of 180 rpm in orbital
shaker.
At the end of 1 hour incubation period spread the transformed cells to the petri plates. Then
select the colonies that are positive. Then with these colonies perform Miniprep for screening.
Mini prep for screening
1) Choose the positive colony and carry out MiniPrep for isolation of the pGEX-2T+Tat
gene.
2) Then double digestion with EcoRI and BamHI to control the presence of Tat gene in the
pGEX-2T vector.
3) Load the digestion mixture on 1% Agarose gel to control and observe the digestion
products.
4) Then isolate Tat gene from the gel with kit and perform PCR to amplify the gene.

References
1) Principles of Gene Manipulation, 6th Edition; Primrose, Twyman and Old.
2) https://www.sigmaaldrich.com/technical-documents/protocols/biology/gst-gene-fusion-
system/pgex-vectors.html
3) https://www.thermofisher.com/tr/en/home/brands/thermo-scientific/molecular-
biology/thermo-scientific-restriction-modifying-enzymes/restriction-enzymes-thermo-
scientific/conventional-restriction-enzymes-thermo-scientific/reaction-conditions-for-
restriction-enzymes.html

43
Experiment-V: Expression analysis of TAT mRNA
Introduction
RNA- Ribonucleic acid
Ribonucleic acid that is so-called RNA, is a polymer comprised of ribonucleotides. RNA
basically works on broad range of roles including translation of genetic information (DNA) into
proteins and regulation of gene activity.The synthesis of RNA molecules from DNA is
performed by RNA polymerases that is further processed by other enzymes. The RNA
synthesized from a protein coding gene then acts as a template for protein synthesis.
When RNA is compared with DNA, it seems that there are several structural details of RNA
which is different than that of DNA. The sugar in DNA is deoxyribose, while RNA contains
ribose sugar that is characterized by the presence of 2’-OH group that makes RNA molecules
less stable compared to DNA. In addition to that, the bases in DNA are Adenine (A), Thymine
(T), Guanine (G) and Cytosine (C), while RNA contains Uracil (U) rather than Thymine (T).
Furthermore, DNA molecules are in the form of double helices containing two strands, whereas
RNA molecules have only one strand that is shorter than DNA strands.
Isolation of RNA
The extraction and purification of RNA molecules from biological samples such as bacteria,
yeast, human blood or tissues, is called as RNA isolation. As ribonuclease enzymes are present
in cells for degrading the RNA molecules, the RNA isolation process is much harder compared
with the DNA isolation methods. The most important approach in the isolation procedure is to
get intact and high quality RNA molecules to be used for further experiments such as cDNA
synthesis, RT-qPCR or RNA mapping.
There are several methods to isolate RNA from biological samples which can be performed
either manually or by commercial kits. In manual methods, phenol- chloroform solutions are
commonly used to remove proteins from the solution by organic phase and purified RNA is
obtained, while in commercial kits, special columns are used to which RNA molecules are
bound and other impurities are removed. The elution of RNA molecules from columns are then
performed by special solutions that make RNA molecules leave the columns.
Overall, in each method, the purified RNA molecules should be resuspended in RNase-free
solution and stored at -800C.
Complementary DNA (cDNA)
Complementary DNA (cDNA) is a double-stranded DNA that is synthesized in the
laboratory from RNA (usually mRNA). This synthesis is performed by Reverse Transcriptase
(RT) enzyme obtained from retroviruses and the single-stranded mRNA molecule is used as a
template for the synthesis of double-stranded DNA that is complementary copy of mRNA
molecule.
The enzyme reverse transcriptase is an RNA-dependent DNA polymerase which transcribes
ss-RNA into DNA. This enzyme works on the formation of ds-DNA when mRNA is reverse
transcribed into ss-cDNA.
Usage of cDNA:
- For nucleotide sequencing and gene expression analysis.

44
 - For production of probes to target chromosomal loci.
- For inserting a fragment of interest into host-vector systems for cloning and/or expression
- For construction of cDNA libraries (collections of random fragments of cDNA which
contain all the mRNA being transcribed by the source cells at the time).
Real – time PCR
Real-time PCR is a technique that measures accumulation of the products that are being
amplified in real time as the reaction continues. Signal coming from the reaction tubes are
quantified for each cycle. Because of quantification component, the method is frequently
referred as Real-Time qPCR. The term RT-PCR is used for PCR reaction using cDNA
synthesized by reverse transcription (RT) as template The technique could be quantitative, in
which an exact quantification is performed based on a standard curve or semi-quantitative, in
which quantification is relative and the comparison is carried out within the samples. In semi-
quantitative real-time qPCR, sequence specific primers for DNA or RNA of interest are used
to determine the relative number of copies which are then used to compare the expression levels
of particular DNA or RNA to a specific housekeeping gene.
The quantification for the amplified product is performed by the use of fluorescent probes
that enable the formation of fluorescence signals with the accumulation of amplified products.
This accumulation gives a detectable fluorescence signal at a specific cycle which is called the
quantification cycle (Ct, Cq). A free-floating fluorescent dye, SYBR Green, generally used to
detect amplified products of Real-Time qPCR reactions. It increases in fluorescence when it
binds to double-stranded DNA.
Melting-curve analysis: The SYBR Green dye binds to double-stranded DNA non-
specifically and non-specific amplified products such as primer dimers can be detected by
analyzing the melting-curves. The melt curve plot (also called a dissociation curve plot)
indicates the melting temperature (Tm) of a target or nonspecific PCR amplification with various
peaks, separately. Therefore, it provides to confirm that target-specific amplification occurred
with the aid of nonspecific nature of SYBR Green as a fluorophore.

Figure 5.1. The melting-curve analysis from a RT-qPCR assay

Each real-time qPCR set-up, negative and positive control samples should be included. A
negative control sample is the sample having no template (DNA or RNA, target molecule) in
order to reveal that there is no intrinsic fluorescence, whereas a positive control sample is the
sample including a housekeeping gene (present and expressed in all cell types) that enables
comparison between samples.
45
Safety
Some components of the NucleoSpin® kit (such as RA1, RA2) contain hazardous contents.
They are harmful by inhalation, in contact with skin, and if swallowed. Wear gloves and
goggles.

Materials and Methods

NucleoSpin® RNA Isolation Kit
First Strand cDNA Synthesis Kit
Sybr Green Real-Time PCR kit

Protocol
The protocol is taken from Macherey-Nagel Total RNA Isolation User Manual.
One of the most important aspects in the isolation of RNA is to prevent degradation of the
RNA during the isolation procedure. With the NucleoSpin® RNA methods, cells are lysed by
incubation in a solution containing large amounts of chaotropic ions. This lysis buffer
immediately inactivates RNases – which are present in virtually all biological materials – and
creates appropriate binding conditions which favor adsorption of RNA to the silica membrane.
Contaminating DNA, which is also bound to the silica membrane, is removed by an rDNase
solution which is directly applied onto the silica membrane during the preparation. Simple
washing steps with two different buffers remove salts, metabolites and macromolecular cellular
components. Pure RNA is finally eluted under low ionic strength conditions with RNase-free
H2O.
Total RNA isolation with NucleoSpin® RNA II kit from bacterial cells
Sample Homogenization
1. Resuspend the bacterial cell pellet (Gram-negative strains) in 100 µL TE buffer (10 mM
Tris-HCl, 1 mM EDTA; pH 8) containing 1 mg / mL lysozyme by vigorous vortexing.
2. Incubate at 37 °C for 10 min.
For preparation of RNA from Gram-positive bacteria, resuspend cells in 100 µL TE
containing 2 mg / mL lysozyme. It may be necessary to optimize incubation time and lysozyme
concentration, depending on the bacterial strain.
Note: Due to the much higher concentration of genome equivalents in a nucleic acid
preparation of bacteria compared with eukaryotic material, it may be necessary to use a lower
quantity of cells for the preparation.
Cell Lysis
- Add 350 µL Buffer RA1 and 3.5 µL ß-mercaptoethanol to the suspension and vortex
vigorously.
For appropriate sample and lysis buffer amounts see Table 2.

46
 Note: As alternative to ß-ME, the reducing agent DTT or TCEP may be used. Use a final
concentration of 10 – 20 mM DTT or TCEP within the Lysis Buffer RA1.

Lysate Filtration
-Reduce viscosity and turbidity of the solution by filtration through NucleoSpin® Filters
(violet rings). Place NucleoSpin® Filters in Collection Tubes (2 mL), apply mixture, and
centrifuge for 1 min at 11,000 x g.
-In case of visible pellet formation (depending on sample amount and nature) transfer
supernatant without any formed pellet to a new 1.5 mL microcentrifuge tube (not supplied).
Alternatively, the lysate may be passed ≥ 5 times through a 0.9 mm needle (20 gauges) fitted to
a syringe.
Adjusting RNA binding conditions
Add 350 µL of ethanol (70 %) to the lysate and mix by vortexing.
RNA Binding
- For each preparation take one NucleoSpin® RNA II Column (light blue ring) placed in a
Collection Tube.
- Pipette lysate up and down 2 – 3 times and load the lysate to the column. Centrifuge for 30
s at 11,000 x g.
- Place the column in a new Collection Tube (2 mL).
- Maximal loading capacity of NucleoSpin® RNA II Columns is 750 µL. Repeat the
procedure if larger volumes are to be processed.

47
 Desalting silica membrane
- Add 350 µL MDB (Membrane Desalting Buffer) and centrifuge at 11,000 x g for 1 min to
dry the membrane.
Salt removal will make the following rDNase digest much more effective. If the column
outlet has come into contact with the flow-through for any reason, discard the flow-through and
centrifuge again for 30 s at 11.000 x g.
DNA Digestion
- Prepare DNase reaction mixture in a sterile 1.5 mL microcentrifuge tube (not provided).
For each isolation, add 10 µL reconstituted rDNase to 90 µL Reaction Buffer for rDNase. Mix
by flicking the tube.
- Apply 95 µL DNase reaction mixture directly onto the center of the silica membrane of the
column. Incubate at room temperature for 15 min.
Wash and dry silica membrane
1st wash
Add 200 µL Buffer RA2 to the NucleoSpin® RNA II Column. Centrifuge for 30 s at 11,000
xg. Place the column into a new Collection Tube (2 mL).
Buffer RA2 will inactivate the rDNase.
2nd wash
Add 600 µL Buffer RA3 to the NucleoSpin® RNA II Column. Centrifuge for 30 s at 11.000
x g. Discard flow-through and place the column back into the Collection Tube.
Note: Make sure that residual buffer from the previous wash step is washed away with Buffer
RA3.
3rd wash
Add 250 µL Buffer RA3 to the NucleoSpin® RNA II Column. Centrifuge for 2 min at 11.000
x g to dry the membrane completely. Place the column into a nuclease-free Collection Tube
(1.5 mL, supplied).
If for any reason, the liquid level in the Collection Tube has reached the NucleoSpin® RNA
II Column after centrifugation, discard flow-through, and centrifuge again.
Note: Make sure that residual buffer from the previous wash step is washed away with Buffer
RA3.
Elution of RNA
- Elute the RNA in 60 µL RNase-free H2O, (supplied) and centrifuge at 11.000 x g. for 1
min.
- If higher RNA concentrations are desired, elution can be done with 40 µL. Overall yield;
however, will decrease when using smaller volumes.

48
 Note: Increased yield for longer templates may be obtained by increasing the concentration
of deoxynucleotides from 500 µM to 1 mM for each dNTP.
5. Incubate the reaction tubes at 25 °C for 15 minutes if using random nonamers. If using
oligo(dT)23 or a specific primer, this step is not needed. This preincubation step allows these
primers to be extended by the enhanced avian RT before incubating at a temperature between
42-50 °C.
6. Place tubes at a temperature between 42-50 °C for 50 minutes.
Note: The optimal reaction temperature should be determined empirically. It is suggested
that the reaction be run at a temperature between 42-50 °C initially. Raising the transcription
reaction temperature incrementally (up to 65 °C) is recommended for transcribing templates
with difficult secondary structure. If the transcription reaction is run at elevated temperatures,
a drop in yield may occur.
7. The first strand cDNA is now ready for subsequent PCR amplification.
Real – Time PCR with DyNAmo HS SYBR green qPCR kit
The protocol is taken from DyNAmo™ HS SYBR® Green qPCR Kit.
- Thaw template DNA, primers and master mix (and ROX passive reference dye if
necessary). Mix the individual solutions to assure homogeneity. This is especially important for
the master mix.
- Prepare a PCR premix by mixing master mix, primers, (ROX) and ddH2O. Mix the PCR
premix thoroughly to assure homogeneity. Dispense appropriate volumes into strip tubes or
plate wells.
- Add template DNA (< 500 ng/50 µl reaction) to the strip tubes or plate wells
containing the PCR premix. For two-step qRT-PCR, the volume of the cDNA added (from the
RT reaction) as the template should not exceed 10% of the final PCR volume.
- Place the strips or plate in the thermal cycler and start the cycling program.

50
cDNA synthesis with Enhanced Avian First Strand Synthesis Kit
The protocol is taken from Enhanced Avian RT First Strand Synthesis Kit.
The optimal conditions for the concentration of Reverse Transcriptase, template RNA, and
primers will depend on the system being utilized and should be determined empirically.
1. Add the following reagents to a thin-walled 200 µl or 500 µl PCR microcentrifuge tube
on ice:
Volume Reagent 3 reactions Final Concentration

In general 0.005- 0.25 µg/µl total RNA or

x µl RNA template 3x µl desired amount of poly (A)+ RNA

1 µl Deoxynucleotide mix 3 µl 500 µM each dNTP

1 µl Random nonamers 3 µl 2.5 µM (In general, use between 1–4 µM)

OR
3’ Antisense specific primer 1 µM (In general,use between 0.5-1 µM)
OR
3.5 µM (In general, use between 1–3,5
Oligo (dT)23 µM)

3x µl q.s. (dependent on the amount of RNA

x µl Water, PCR reagent template)

10 µl Total Volume 30 µl
2. Mix gently and briefly centrifuge to collect all components to the bottom of the tube.
3. Place tube in the thermal cycler at 70 °C for 10 minutes.
Note: This 70 °C incubation step before the reverse transcription reaction is optional. This
step may denature RNA secondary structure, which will allow for more efficient reverse
transcription. All of the reverse transcription components may be added together in one tube
and placed immediately at the optimal reverse transcription temperature unless random primers
are being used. If using random nanomers, 15 minute incubation at 25 °C is required.
4. Remove tube, place on ice, centrifuge and add the following components to the reaction:

Volume Reagent 3 reaction Final Concentration

2 µl 10X buffer for RT (Reverse Transcriptase) 6 µl 1X

1 µl RT (Reverse Transcriptase) 3 µl 1 unit/µl

1 µl RNase inhibitor 3 µl 1 unit/µl

6 µl Water 18 µl …

20 µl Total volume 60 µl

49
References

1.https://www.bio-rad.com/en-uk/applications-technologies/what-real-time-pcr-
qpcr?ID=LUSO4W8UU
2. Tan SC., Yiap BC. DNA, RNA and Protein Extraction: The Past and The Present. J
Biomed Biotechnol. 2009; 2009: 574398.
3. Molecular Biology of the Cell. 4th edition.
4.https://www.ebi.ac.uk/training/online/course/biomacromolecular-structures-introduction-
ebi-reso/rna
5. Enhanced Avian RT First Strand Synthesis Kit
6. DyNAmo™ HS SYBR® Green qPCR Kit
7. Macherey-Nagel Total RNA Isolation User Manual

51