DNA Cloning
Introduction and Idea
The word "cloning" can be used in many ways. In this document, it refers to making
multiple, exact copies of a particular sequence of DNA. To make a clone, a target DNA
sequence is inserted into what is called a cloning vector. A cloning vector is a DNA
molecule originating from a virus, plasmid, or the cell of a higher organism into which
another DNA fragment of appropriate size can be integrated without interfering with the
vector's capacity for self-replication. The target and vector DNA fragments are then
ligated, or joined together, to create what is called a recombinant DNA molecule.
Recombinant DNA molecules are usually introduced into Escherichia coli, or E. coli—a
common laboratory strain of a bacterium— by transformation, the natural DNA uptake
mechanism possessed by bacteria. Within the bacterium, the vector directs the
multiplication of the recombinant DNA molecule, producing a number of identical
copies. The vector replication process is such that only one recombinant DNA molecule
can propagate within a single bacterium; therefore, each resulting clone contains multiple
copies of just one DNA insert. The DNA can then be isolated using the techniques
described earlier.
A restriction enzyme is a protein that binds to a DNA molecule at a specific sequence
and makes a double-stranded cut at, or near, that sequence. Restriction enzymes have
specialized applications in various scientific techniques, such as manipulating DNA
molecules during cloning. These enzymes can cut DNA in two different ways. Many
make a simple double-stranded cut, giving a sequence what are called blunt or flush
ends. Others cut the two DNA strands at different positions, usually just a few
nucleotides apart, such that the resulting DNA fragments have short single-stranded
overhangs, called sticky or cohesive ends. By carefully choosing the appropriate
restriction enzymes, a researcher can cut out a target DNA sequence, open up a cloning
vector, and join the two DNA fragments to form a recombinant DNA molecule.
In general, a bacterial genome consists of a single, circular chromosome. They can also
contain much smaller extrachromosomal genetic elements, called plasmids, that are
distinct from the normal bacterial genome and are nonessential for cell survival under
normal conditions. Plasmids are capable of copying themselves independently of the
chromosome and can easily move from one bacterium to another. In addition, some
plasmids are capable of integrating into a host genome. This makes them an excellent
vehicle, or vector, for shuttling target DNA into a bacterial host. By cutting both the
target and plasmid DNA with the same restriction enzyme, complementary base pairs are
formed on each DNA fragment. These fragments may then be joined together, creating a
new circular plasmid that contains the target DNA. This recombinant plasmid is then
coaxed into a bacterial host where it is copied, or replicated, as though it were a normal
plasmid.
Bacterial plasmids were the first vectors used to transfer genetic information and are
still used extensively. However, their use is sometimes limited by the amount of target
DNA they can accept, approximately 15,000 bases, or 15 Kb. With DNA sequences
beyond this size, the efficiency of the vector decreases because it now has trouble
entering the cell and replicating itself. However, other vectors have been discovered or
created that can accept larger target DNA including: bacteriophages, bacterial viruses
that accept inserts up to 20 Kb; cosmids, recombinant plasmids with bacteriophage
components that accept inserts up to 45 Kb; bacterial artificial chromosomes (BACs)
that accept inserts up to 150 Kb; and yeast artificial chromosomes (YACs) that accept
inserts up to 1000 kb. Many viruses have also been modified for use as cloning vectors.
1. Protocol
2. Reagents needed :



ampiceline stock : 25 mg/ml in destilled water (filter sterilization)
(selection for E. coli with vector in it.)
tetracycline stock : 12,5 mg/ml in destilled water (no sterilization
required) (light sensitive)
Xgal stock : 2 % in dimethylformamide (no sterilization required) (light
sensitive)
IPTG stock : 1 M in destilled water (filter sterilization)
glycerol 60 % (v/v) : (heat sterilization)
o
Ca/Mg solution : 75 mM CaCl2 and 25 mM MgCl2 (heat sterilization)


o
o
LB medium : L-Broth medium
primers :
 M13L : 5' - CGC CAG GGT TTT CCC AGT CAC GAC - 3'
 M13R : 5' - AGC GGA TAA CAA TTT CAC ACA GGA - 3'
3. Preparation of competent E. coli cells :
1. Inoculate 1 single colony of E. coli in 5 ml L-Broth medium and grow
overnight at 37°C in a shaking incubator.
 (strain XL 1 Blue : add 3 µl tetracycline to the 5 ml LB medium)
 (strain DH5a : no additives in medium !!)
2. Bring 20 µl of this overnight culture in 100 ml LB medium. Grow the
culture while shaking at 37°C until OD550 = 0.3 to 0.5. (THIS OD IS
CRITICAL !!!) (this takes 5 to 9 hours) (for faster growth : inoculate up to
1 ml culture in 100 ml medium)
 (strain XL 1 Blue : add 60 µl tetracycline to the LB medium)
 (strain DH5a : no additives in medium !!)
3. Cool the cultures on ice for 10 minutes. Collect the bacteria by
centrifugation at 4.000 rpm for 10 minutes. Resuspend the pellet in 50 ml
Ca/Mg solution. Incubate 30 minutes on ice.
4. Collect the bacteria by centrifugation at 4.000 rpm for 10 minutes.
Resuspend the pellet in 10 ml Ca/Mg solution. Incubate 15 minutes on ice.
5. Add 3,4 ml sterile (60 %) glycerol to the 10 ml culture (endconcentration
glycerol = 15 %) and resuspend. Devide the suspension into aliquots of
300 µl (in 1,5 ml tubes). Store at -70°C.
4. Cleaning of PCR product : use High Pure PCR product purification kit
(Boehringer).
If the band on gel is weak, concentrate the product by using 50 µl for cleaning on
column and resuspend it in 20 µl.
5. Ligation : use pGEM-T and pGEM-T Easy Vector Systems (Promega).
0. Set up ligation reactions as below :
 2x Rapid Ligation Buffer : 5 µl
 pGEM-T vector : 1 µl
 T4 DNA ligase : 1 µl
 PCR product : 3 µl
1. Mix and incubate overnight at 4°C
6. Transformation :
0.
1.
2.
3.
4.
5.
6.
Put a frozen tube with competent cells on ice until thawed
Add 2 µl of the ligate on the cells and mix gently
Place on ice for 30 minutes
Heat-shock the cells for 1,5 minute at EXACTLY 42°C
Put on ice for 2 minutes
add 1 ml L-Broth medium to the tube and incubate 1 hour at 37°C
Spin the cells down and discard most of the supernatans : leave 100 µl in
the tube
7. Prepare agar plates :
 (for XL1 Blue : add ampiceline (2 µl stock/ml agar) and
tetracycline (1,2 µl stock/ml agar) to the agar (cooled to 50°C)
before pooring)
 (for DH5a : add ampiceline (2 µl stock/ml agar) to the agar (cooled
to 50°C) before pooring)
bring 87 µl Xgal (2 %) and 2 µl IPTG (1M) on the plate and spread
8. Suspend the cells, bring the 100 µl suspension on the plate and spread
9. Incubate overnight at 37°C
10. The white colonies are positive
If you need to make plasmid DNA, perform 7. in stead of 6.
7. PCR of the positive colonies :
0. Pick a white colony with a toothpick and rub in in a PCR tube, repeat for
several colonies
1. Add PCR mixture to the tube and start the PCR
2. Check the size of the PCR product on gel
3. The fragments of the right size can be sequenced
8. PCR of the positive colonies and plasmid isolation:
0. Pick a white colony with a toothpick, rub in in a PCR tube and streak the
cells also on a L-Broth agar plate (1 cm streak for each colony. When you
mark the outside of the plate with a grid, you can inoculate up to 50
colonies on 1 plate). Repeat for several colonies. Incubate the plate at
37°C.
1. Add PCR mixture to the tube and start the PCR.
2. Check the size of the PCR product on gel for the right fragments.
3. During the PCR, the streaks from the positive colonies are grown.
Inoculate the streaks, that gave an expected band in PCR, in 5 ml L-Broth
and incubate at 37°C overnight while shaking.
4. Spin down the bacteria (2 times 1,5 ml) for 2 minutes at 6000 rpm.
5. Prepare plasmide with the kit : High Pure Plasmid Isolation Kit from
Boehringer.
DNA Blotting
Introduction and Idea:
The porous and thin nature of a gel is ideal for separating DNA fragments using
electrophoresis, but as we mentioned earlier, these gels are delicate and rarely usable for
other techniques. For this reason, DNA that has been separated by electrophoresis is
transferred from a gel to an easy-to-handle inert membrane, a process called blotting. The
term "blotting" describes the overlaying of the membrane on the gel and the application
of a pad to ensure even contact, without disturbing the positions of the DNA fragments.
In the first step, the DNA trapped in the gel is denatured—the double-stranded DNA is
broken into single strands by soaking the gel in an alkaline solution. This readies the
DNA for hybridization with a probe, a piece of DNA that is complementary to the
sequence under investigation. A membrane, usually made of a compound called
nitrocellulose, is then placed on top of the gel and compressed with a heavy weight. The
DNA is transferred from the gel to the membrane by simple capillary action. This
procedure reproduces the exact pattern of DNA captured in the gel on the membrane. The
membrane can then be probed with a DNA marker to verify the presence of a target
sequence. Southern Blotting
1.
Electrophoresis of DNA is carried out in a neutral agarose gel system. Prepare a
0.8-1% agarose gel containing 1x TAE buffer. Ethidium bromide can be added to a final
concentration of 0.2 µg/ml.
2.
Apply the samples to the gel.
3.
Run the gel in 1x TAE. buffer at 4V/cm until the bromophenol blue indicates that
the sample has run for a sufficient distance.
4.
Following electrophoresis, visualize the gel under UV transillumination and
photograph with a ruler.
5.
i) Depurination, 10 minutes at room temperature with gentle agitation (optional). This
step
ii)
is
necessary
Denaturation,
if
25
target
minutes
sequences
at
room
are
greater
temperature
than
with
10
Kb
gentle
in
size
agitation.
iii) Neutralization, 30 minutes at room temperature with gentle agitation. When using
nitrocellulose membranes, the neutralization time should be extended to 45 minutes.
Include a rinse in distilled water between each step
6.
Assemble the capillary blotting apparatus using 10X SSC as the transfer buffer.
Allow the DNA to transfer overnight onto Hybond N+.
7.
The following day, disassemble the apparatus, mark the membrane appropriately
and fix the DNA to the membrane by UV crosslinking or baking (2 hours at 80°C). For
nitrocellulose membranes, bake for 2 hrs. at 80°C in a vacuum oven.
Solutions:
Hybridization buffer
5x SSC
1 in 20 dilution Liquid Block (Amersham) or other blocking reagent
0.1%(w/v) SDS
5%(w/v) Dextran sulphate
EDTA stock
0.5M EDTA pH8.0
SDS stock
10% or 20% (w/v) SDS
Depurination solution
250mM HCl
Denaturation solution
1.5M NaCl
0.5M NaOH
Neutralization solution
1.5M NaCl
0.5M Tris-HCl
pH adjusted to 7.5
20x SSC
0.3MNa(3)citrate
3M NaCl
Dot blot hybridization
Blotting of the sample
1. Take 20ul of the PCR product and cDNA in to tube and denature with 3ul
(10mM) EDTA, 3ul (0.4M) NaOH and 4ul H2O.
2. Boil the mixure for 10 min; neutralize using equal vol. (30ul) of 2M ammonium
acetate, PH 7.
3. Fit the membrane to the blotter and cut the exact size to fit the manifold,
4. Tight the screw in diagonal crossing.
5. Immerse the membrane in SC and the apply the vacuum.
6. Blot Each sample and the remve the membrane and neutralize by washing in 2X
SSC for 30-60 sand then in 0.4 NaOH for 30-60s .and then in neutralizing
solution (1M Tris HCL ,1.5 M NaCl ,PH 8)for 1s
7. fix the DNA by exposure to UV (5 min) and air-dry.
Kinasing of oligos (Probes). Invitrogen
1
10X kinase buffer
1µl
2
PCR product
2 µl
3
32P ATP
l5 µl
4
water
1 µl
5
T4 poly kinase
1 µl
Total volume 10 ul
Incubate at 370C for 30 minutes. Store at 4C.
Purification of labeled probe:
Method I (using ProbeQuant G-50 Micro column):
1. Resuspend the resin in the column by vortexing.
2. Loosen the cap one-fourth turn and snap off the bottom closure.
3. Place the column in a 1.5 eppendorf tube and pre-spin the column for 1 minute at
3000 rpm.
4. Place the column in a new 1.5 ml tube and slowly apply 50l (10l labeled probe
plus 40l STE) to the top center of the resin carefully.
5. Spin the column at 3000rpm for 2 minutes. The purified sample collects at the bottom
of the eppendorf tube.
6. Method II (reference Maniatis;purification of labeled oligo spin columns)
Hybridization of probe:
Prehybridization:
1 hr at 55oC in hybridization oven
6XSSC
0.5% Sodium pyrophosphate
5X Denhardt’s solution
100g/ml denatured salmon sperm DNA
Hybridization:
1 hr at 55oC in hybridization oven
6XSSC
0.5% Sodium pyrophosphate
5X Denhardt’s solution
10% Dextran sulphate (optional)
50g/ml denatured salmon sperm DNA
Pour off the pre-hybridization mix and add labeled probe: 106 cpm /ml.
Washing:
Wash the membrane twice with 6XSSC containing 0.5% sodium pyrophosphate for 30
minutes 55oC.
Air-dry the membrane and autoradiograph.