MOLECULAR CLONING
Rüveyda AKÇİN, Gebze Technical University, Turkey
AIM
To integrate the desired pAc 3.5 gene to pUC19 with the T4 ligase.
INTRODUCTION
The process placing of a gene to vector and
forming many copies of the gene it contains
during the division of cells as a result of
transformation of this vector into bacterial cells
is called "gene cloning". (Figure 1) Cloning is
often used to advance gene-containing DNA
fragments.
Figure 1
Important points in gene cloning:
1. Purification of gene bearing DNA (and
RNA)
2. Determination of gene location
3. Remove of gene
4. Obtaining of carrier DNA
5. Coupling of gene DNA and vector DNA
6. Transferring the recombinant vector to
the cell
7. Selection
8. Control of gene product
The gene to be cloned and the plasmid are
combined with the T4 DNA ligase enzyme (T4
closes gaps formed in double-stranded DNA. It
is an enzyme encoded by the T4 phage.) under
appropriate heat and reaction conditions. The
temperature can vary from + 4 ° C to + 25 ° C,
and the incubation time can vary from 5
minutes to one night. The T4 DNA ligase
enzyme from the T4 bacteriophages is derived
from the E. coli DNA ligase enzyme 400 times
more effective. The T4 DNA ligase uses ATP as
the cofactor. For this reason, T4 DNA Ligase
buffers containing ATP should not very
frequently be frozen and thawed. T4 is the only
ligase that effectively combines blunt ends in
normal reaction conditions. The vector and
insert DNA ratio must be calculated for the
ligation of vector and insert DNA. Calculated as
follows:
Inser mass in nanogram (ng) = [(ng of vector x
size of insert (in kb)) / size of vector (in kb)] x
(insert : vector molar ratio)
MATERIALS
 Cloning vector (digested with appropriate
REs)
pUC19
 Insert DNA (digested with the same REs as
vector)
pAc 3.5
 T4 DNA Ligase (1 U/µl)
 T4 DNA Ligase Buffer (10X)
400 mM Tris-HCl, 100 mM MgCl2, 100 mM
DTT, 5 mM ATP (pH 7.8 at 25°C)
 ATP (10 mM)
The T4 DNA ligase uses ATP as the cofactor.
METHOD
Ligation Tube
Components RXN 1 (İnsert+Vector) RXN 2
Vector (0.075 g/l) 1 l
Insert (0.1 g/l ) 2.9 l
T4 DNA Ligase Buffer (10X) 3 l
ATP (10mM) 1.5 l
dH2O 21.1l
T4 DNA Ligase (1 U/l) 0.5 l
Total Volume 30l
1. Combine the materials (except T4 DNA
Ligase) given in the table above in a 1.5 ml
eppendorf tube (on ice). Prepare a control
reaction with no insert and add 5.8 l
sterile water instead of insert.
PREPERATION of COMPETENT CELLS
AIM
To prepare competent cells for transformation.
INTRODUCTION
Competent cells are required for
transformation. These are the hosts. There are
many different types. The important point is
the presence of the resistance genes in these.
The most commonly used competent cells are
C600, RR1, JM101, HB101, NM539, LE392,
JM101, Y1089, JM109, Y1090. They are stored
in freezer (-20 / -70 ° C) in glycerol.
There are various methods for preparing
compete cells. In both Ca+2 and electroporation
methods, firstly the cell must be made
competent. Leads to the precipitation of CaCl2
DNA on the outside of the bacteria or in some
way, it makes some changes in the cell wall,
thereby increasing DNA binding. The DNA
enters the bacteria with an increase in ambient
2. Gently mix the tubes and after a short spin
incubate them at 37 C for 10 minutes.
Control Tube 3. Put the tubes in ice-water bath (0 C) and
wait for 10 minutes. (Because the ligation
1 l takes place in an aqueous environment.)
- 4. Add 1 l T4 DNA Ligase (1 U/l) to each
3 l reaction tube and mix gently. After a short
1.5 l spin put the tubes in ice-water bath again
24 l and wait for 30 minutes. (T4 DNA Ligase
0.5 l should be added after the first incubation
30 l at 0 C)
5. Place the tubes in water bath adjusted to
16 C and incubate them overnight.
(It can also be kept at room temperature.)
6. Ligation reaction is stopped by incubating
the tubes at 70 C for 10 minutes.
7. After a short spin, ligation mixtures should
be used immediately for transformation or
can be stored at 20 C for a few days.
temperature of 42 ° C. It is not clear why heat
shock is effective, but it is thought that it allows
the DNA of the plasma membrane to pass
through the loosened poros. In another
method, the transformation storage solution
(TSS) buffer method, competence is induced by
polyethylene glycol (PEG). This technique is
relatively simple and does not require heat
shock.
MATERİALS
TSS (Transformation and Storage Solution for
Chemical Transformation):
 LB (Tryptone 1 %, Yeast Extract 0.5 %, NaCI
1 %)
 Luria-Bertani (LB) broth is a rich medium
that permits fast growth and good growth
yields for many species including E. Coli. LB
can support E. coli growth (OD600=2–3)
under normal shaking incubation
conditions.
 % 10 PEG 8000 (W/V)
it helps in shielding the negative charges
present on the DNA and host cell
membrane.
 % 5 DMSO (V/V)
DMSO brings together the reagents that
have been added during the reaction.
 50 mM MgCI2 pH 6.5
MgCl2 acts in the same way as does CaCl2.
It induces the ability of the cells to take up
DNA by altering the permeability of the
membranes.
This solution can be stored at 4 C for two week
after autoclaved.
METHOD
1. Streak the cells from -20 C stock on a LB
plate. Incubate the pplate at 37 C
overnigth.
2. Pick a single, well-isolated colony and
inoculate it into 5 ml of LB broth. Incubate
at 37 C overnigt with shaking at 200 rpm.
BACTERIAL TRANSFORMATION
AIM
To transfer the conjugate vector pAc 3.5 and pUC19 to the E. coli JM 109
INTRODUCTION
Transformation in molecular biology means the
introduction of a foreign gene into a cell and it
3. Transfer 1 ml of the saturated overnight
culture to a sterile 500 ml flask containing
100 ml of LB medium (do not add antibiotic
at this step). Incubate the cells at 37 C
with shaking at 200 rpm (Seperated two
tubes and take 800 µl in medium.), until
OD600 reach 0.5; this usually takes 2.0-2.5
hr. Check the OD frequently when it gets
beyond 0.2 to avoid overgrowth.
(OD600 is an abbreviation indicating the
absorbance, or optical density, of a sample
measured at a wavelength of 600 nm. It is
a common method for estimating the
concentration of bacterial or other cells in
a liquid.)
4. When the culture reaches an OD600 of 0.5,
chill the flask on ice for 20 min and then
collect the cells by centrifugation at 700
rpm for 10 min at 4 C.
5. Pour off supernant and resuspend each
pellet in 10 ml of ice-cold TSS solution. Now
the competent cells are ready to be
transformed. (Then seperated 20 tubes.)
6. If they are not immediately used, cells can
be stored at 4 C for maximum of 6 hr
without significant loss of competency. The
same competent cells can also be stored at
-70 C for long-term storage.
Transformation frequency of frozen cells is
30 % of that of the fresh cells, when used
within two months.
is part of the DNA genome so, the cell changes.
Transformation of plasmid DNA into E. coli
using the heat shock method is a basic
technique of molecular biology. It consists of
inserting a foreign plasmid or ligation product
into bacteria. In a bacterial cell population, cells
with a specific physical property, called only
recipient competent cells, take in this DNA.
DNA enters from the receptor sites on the
surface of the bacterial cell. During this
transition energy and special transport
molecules are required.
To distinguish the few transformed cells from
the millions of untransformed ones, antibiotic
(Ampicillinis used this experiment) selection is
used.
Positive Control: Transform competent cells
with plasmid DNA (not digested), provides
measure of the efficiency of transformation
and serves as a standard for comparison with
other transformations.
Negative Controls: No DNA; transform
competent cells, without added DNA, and plate
on selection media; if high background, may
indicate a problem with the antibiotic in the
agar plates. The figure below shows the
general logic of transformation;
MATERİALS
 Competent cells (E. Coli JM 109)
 Ligation materials (from the molecular
cloning experiment)
- RXN 1 (insert + vector)( pUC19 plasmid
vector + insert DNA; both of which
were digested with the same
restriction enzymes)
- RXN 2 (vector)( pUC19 plasmid vector
which was digested with the same
restiction enzymes as insert DNA)
(Negative Control)
 pUC19 plasmid DNA (nondigested) (will
be used as positive control)
(JM109 is a K strain bacterium that
provides minimized recombination and aids
in plasmid stability which results in high
quality plasmid DNA preparation. The
strain carries the hsdR17 genotype, which
prevents cleavage of heterologous DNA by
an endogenous endonuclease. JM109
strain supports Alpha-complementation for
blue/white screening for recombinant
plasmids.)
METHOD
1. Firstly, take the competent cells from -70
C freezer and thaw on ice.
2. Pipette 5 µl of each ligation material into
1.5 ml epp tubes separately and place on
ice.
3. Put 1; 10; 25; 50 and 100 ng of pUC19
plasmid DNA (nondigested) into different
1.5 ml epp tubes and place on ice.
LIGATION LIGATION CONTROL NEGATIVE CONTROL POSITIVE CONTROL
12 Groups 2 Groups 1 Group 5 Groups
5 l RXN 1 5 l RXN2 E. Coli JM 109 pUC19 nondigested
100 l LB 100 l LB 100 l LB 100 l LB
Antibiotic + + + +
4. Add 100 µlcompetent cells, thawed on ice,
into each epp tube containing DNA.
Prepare also a control tube containing
competent cells only (The control tube
should br prepared firstly).
5. Gently swirl tubes to mix, then place on ice
for 30 mins.
6. Heat shock cells by placing the tubes into a
42 C water bath for 2 min without shaking.
(Heat shocking causes DNA to enter the
cell.)
7. Put the tubes on ice for 2 min immediately.
8. Add 1 ml LB medium (RT) to each tube.
9. Place the tubes on a shaker at 200 rpm for
1 hr at 37 C. (As the E. coli multiplies at 37
 C, the antibiotic resistance gene Figure 1
increases.)
10. Spin the epp tubes for 30 seconds and
decent the supernatant.
11. Resuspend the pellet in the remaining LB
medium (100 µl) and spread onto
LB/ampicillin containing plates.

(300 ml LB agar, 10 mg/ml ampicillin and final

concentration shoul be 100 µg/ml. How much
ampicillin should be used?

M1xV1=M2xV2

300 ml x 100 µg/ml = 10 mg/ml x a Figure 2

a= 3 ml ampicillin) Ligation Control:
12. Incubate the plates overnight (12 to 16 hr)
at 37 C.
13. Next day morning take the petri plates,
count the colonies and evaluate the result.

RESULT
DNA isolation from agarose gel of nanodrop
result (last week):

pAc 3.5 pUC19

280 0.082 0.134
260 0.103 0.021
4 ng/l 12 ng/l
Figure 3

Ligation:

Figure 4

Negative control:
 Figure 5 Figure 8 (25 ng/µl)
Positive Control:

Figure 6 (1 ng/µl)
Figure 9 (50 ng/µl)

Figure 7 (10 ng/µl) Figure 10 (100 ng/µl)

 CONCLUSIONS
Colony numbers: In the first instance, last week the DNA was
isolated of agarose and measurement was
1 ng → 43
taken with nanodrop. These values are in the
10 ng → 180 result part. According to this results, values are
very low. Under normal conditions the
25 ng → 880
experiment can not be continued with these
50 ng → 892 values. However, if you look at the reason the
error may have been due to previous
100 ng → 1300 experiments. Namely, In PCR may have been
When counting, the petri dish is first divided contaminated or it may be a problem caused by
into 4 pieces. A piece is counted of petri dishes restriction enzymes digestion experiment.
and then, that piece is multiplied by four. There may have been a wrong in the method
steps.
Transformation efficiency =
Secondly, in this experimets the pAc 3.5 and
The number of transformant colonies counted pUC19 digested with restriction enzymes were
on the plate / The amount of DNA transformed combined. Then, the competent cell was
(g) prepared. The vector was introduced into E.coli
JM 109, a competent cell, by the TSS method.
The amount of DNA transformed (g) = The
After that, As you can see in the conclusions,
amount of DNA transformed (ng) x 10-3
the experiment was controlled. and
The following calculations are made according calculations were made according to this.
to the above form. There seems to be no problem in the ligation
results. Perhaps the second figure might have
For 1 ng:
been a little contaminant. (Must be examined
1 ng x 10-3 = 0.001 g in detail.) There is a problem with figure four.
Normally the colony is not observed. Vectors
43 / 0.001 = 43000 g have antibiotic resistance. This state indicates
For 10 ng: that the vector is self-ligating. Purpose of
negative control exposes the target cell is not
10 ng x 10-3 = 0.01 g resistant to antibiotics. However, negative
control has colonies. Something may be wrong
180 / 0.01 = 18000 g
with the antibiotics. Finally, In positive control,
For 25 ng: growing of the untransformed bacterial cells
(pUC19 nondigested) should also be tested on
25 ng x 10-3 = 0.025 g
non-antibiotic plate. As the amount increased,
880 / 0.025 = 35200 g the number of colonies increased in positive
controls. Namely, the value of the
For 50 ng: transformation efficiency increased as the
50 ng x 10-3 = 0.05 g amount increased. This results are correct.
Thus, it can be said that the pUC19 plasmid
892 / 0.05 = 17840 g works correctly.
For 100 ng:

100 ng x 10-3 = 0.1 g

1300 / 0.1 = 13000 g

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