Molecular Biology Lab 13
Cloning PCR Products
Background:
The molecular cloning of DNA fragments into plasmid vectors is a relatively
straightforward process. The DNA molecule can be cleaved with one or more restriction
enzymes to isolate the sequence of interest (insert). Alternately, PCR can be used to
produce a product for cloning. This product is ligated into a vector that has been
linearized by digestion with the same restriction enzyme(s). The products of the ligation
reaction are then used to transform an appropriate strain of E coli. The resulting
transformed colonies are screened by hybridization, PCR, or restriction digest to identify
those that carry the proper insert. There are several different methods or strategies that
are used to clone DNA fragments.
Cloning fragments with protruding ends: One of the easiest methods is to use inserts and
vectors with protruding (sticky) ends. These single stranded ends are easily produced by
digestion with restriction endonucleases or by incorporating a restriction site into your
primers for PCR. The protruding ends anneal to form a hybrid molecule in 2 distinct
steps. First, an intermolecular reaction occurs between one end of the vector and insert to
generate a linear molecule. Second, an intramolecular reaction occurs between the 2 free
ends of each linear DNA to generate a circular molecule. DNA ligase then joins the
annealed strands to form a covalent link. The insert may have 2 orientations relative to
the vector (forward and reverse) and this orientation can be critical. For example, when
cloning into an expression vector, the gene may be expressed as sense or antisense,
depending on the orientation relative to the promoter in the vector.
Other, less desirable products can occur during molecular cloning and these may lead to
problems. For example, cloning can produce linear and circular homo and
heteropolymers of various sizes, orientations, and compositions. The intermolecular
reaction requires high concentrations of both DNAs to maximize joining of the 2 DNAs.
However, the second intramolecular process works best with very low concentrations of
DNA. Low DNA concentrations favor self ligation rather than another intermolecular
reaction. Usually, equimolar amounts of insert and vector are used and the total DNA
concentration is kept to less than 10 g/ml.
Directional cloning: One way to increase the yield of circular monomeric recombinants is
to use a cloning strategy in which the termini are not all equivalent. For example, one can
digest the different ends of the insert and the vector with 2 different enzymes. In this
case, the termini can only ligate in one direction. If you carefully plan which enzymes to
use, you can avoid problems of improper orientation.
Blunt-ended cloning: Fragments of DNA with blunt-ended termini can be cloned into
vectors with blunt ends. However, this is a very inefficient reaction and it requires high
concentrations of DNA ligase and DNA. The first intermolecular reaction is performed at
high DNA concentration, whereas the second intramolecular reaction must be performed
at low concentrations to be effective (to avoid polymers). Usually, the first reaction is
performed at high DNA concentration, the reaction is diluted 20-fold, and the second
reaction is performed.
Blunt-ended DNA fragments can be cloned more efficiently using synthetic linkers or
adaptors purchased from biotech companies. A linker is a short stretch of DNA that
contains a restriction enzyme recognition sequence. These are available for many
restriction sites. They are ligated to the insert using very high concentrations of ligase and
linker. They are then digested with the appropriate enzyme to generate sticky ends.
Following this, they are ligated to a vector that has been predigested with the same
enzyme. Adaptors are similar, however, they are purchased as ‘ready to ligate’ DNA
fragments with one sticky terminus (which can be ligated to vector) and one blunt end
(which is ligated to the insert). The linkers or adaptors often contain sites for restriction
enzymes that cut very infrequently in mammalian DNA (NotI and SalI cut once every
100 – 1000 kb). This makes it unlikely that the added restriction site is also present in the
vector or insert (which would create problems if one were interested in cutting out the
intact insert).
Cloning PCR products: Many methods have been devised for cloning products of PCR
reactions. One problem is that the Taq polymerase used for PCR has a template
independent terminal transferase activity. It adds a single unpaired nucleotide to the end
of the PCR product and this can inhibit cloning. Several useful methods include:
1. Cloning blunt-ended PCR products. Some thermostabile DNA polymerases (Pwo
and Pfu) lack terminal transferase activity and generate suitable blunt end fragments
for cloning. This method is much less efficient than others.
2. Addition of restriction sites to the 5’ termini of primers. These sites are transferred to
the PCR products during amplification. If you use 2 different sites (one on each
primer) you can clone directionally. This method is much more efficient.
3. T vectors contain a single thymidine at each 3’ end. They are used for cloning PCR
products that have a single adenine at the end (caused by the terminal transferase
activity of Taq). A single unpaired T and A are enough to stabilize the strands and
this leads to a cloning efficiency 50X greater than blunt end cloning.
TOPO TA Cloning System: This is a commercially available kit that will be used in this
lab. It uses a Taq polymerase to generate PCR fragments with an A overhang at each end.
The kit comes with a linearized vector that contains single overhangs of T at each end.
No DNA ligase is necessary to join fragments. The linear vector comes with a
topoisomerase molecule attached. Topoisomerases are enzymes that cleave the
phosphodiester backbone in one DNA strand. The energy from the broken bond is
conserved by formation of a covalent bond between the 3’ phosphate and the
topoisomerase enzyme.
This bond between enzyme and the 3’ phosphate can be attacked by the 5’ hydroxyl of
another DNA strand, reversing the reaction and releasing the topoisomerase. This results
in covalent attachment of insert and vector DNA without the need for DNA ligase.
Objectives:
The objectives of this lab are (1) to learn about the different methods for cloning PCR
products and (2) to clone PCR products in lab using the TOPO TA Cloning method.
Materials:
TOPO TA cloning kit (InVitrogen Corp.) containing T vector and other components
required for cloning including the pCR2.1-TOPO vector, 10X PCR buffer, salt solution,
-T1 Competent cells (1
vial/transformation), SOC medium.
Samples of cellular DNA
PUC19 control DNA
37C shaking incubator
42C water bath
LB agar p
X-gal (40 mg/ml)
Spreaders for bacteria
Disposable gloves
Microcentrifuge
Thermocycler
Thin wall PCR tubes
PCR primers
PCR supermix or PCR reaction components
Miniprep kit for DNA purification
Micropipetters
Sterile eppendorf tubes, yellow tips, and blue tips, eppendorf racks
Sterile deionized water
Glycerol and cryotubes for making stocks of overnight cultures
LB broth and antibiotic for overnight cultures
Methods:
PCR reaction.
1. Set up a PCR reaction using high molecular weight cell DNA. Add approximately
1 g of DNA, 2 l of each primer pair, and 45 l of PCR supermix to a PCR tube
on ice. Mix components and place in thermocycler. As a positive control, use 1 l
of control template DNA from the kit along with 1 l of each primer. Amplify
under the following conditions: denaturation at 94ºC for 2 min, then 30 cycles of
94ºC denaturation for 1 min, annealing at 55ºC for 1 min, and extension at 72ºC
for 1 min.
2. Program the thermocycler for the proper PCR reaction conditions, and run
samples to amplify template DNA. Be sure to include a 7 to 30 min extension at
72ºC after the last cycle to ensure that all PCR products are full length and 3’
adenylated
3. Pour a 1.4% agarose gel containing 0.5 g/ml ethidium bromide in 1X TAE
buffer. Allow to polymerize for at least 30 min.
4. Add 10 to 20 l of your PCR reaction to an eppendorf tube and mix with blue
juice gel loading buffer (4 l/20 l of DNA solution). Run agarose minigel at 60V
to visualize the DNA bands. You should obtain a single band of PCR product.
Set up the TOPO TA cloning reaction.
5. Equilibrate a water bath to 42ºC
6. Warm a vial of SOC medium to room temperature and the agar plates to 37°C
temp. for 30 min. Use 2 plates for each transformation.
7. Spread 40 l of 40 mg/ml X-gal on each LB plate and incubate at 37°C until
ready for use.
8. Thaw the competent cells (DH5-T1) on ice. Do not place at 37ºC.
9. Add 0.5 to 4 l of fresh PCR product, 1 l of salt solution, and bring the volume
to 5 l with sterile deionized water. Add 1 l of TOPO vector to the 5 l reaction
and mix gently. Include a positive and negative control (see below).
reagent
fresh PCR product
control PCR product
salt solution
sterile water
TOPO vector
final volume
experimental
0.5 – 4 l
1 l
up to 5 l
1 l
6 l
Pos. control
1 l
1 l
3 l
1 l
6 l
Neg control
1 l
4 l
1 l
6 l
10. Incubate the cloning reactions. For some purposes, 5 min at room temperature is
sufficient. Increasing the time of reaction to 30 min will result in more colonies.
11. Place the reaction on ice. You can store the TOPO cloning reaction overnight at
-20ºC, or you can proceed immediately to the transformation of E coli.
12. Add 2 l of the TOPO cloning reaction into a vial of competent cells and mix
very gently by swirling the yellow tip. Do not pipette up and down to mix.
13. Incubate on ice for 30 min.
14. Heat shock the cells for 30 seconds at 42ºC without shaking.
15. Immediately transfer the tubes to ice.
16. Add 250 l of SOC medium at room temp. Cap the tube and shake for 1 hour at
37ºC in a shaker incubator (200 rpm).
17. Spread 10 to 50 l from each transformation on a prewarmed selective plate and
incubate overnight at 37ºC. Use ampicillin plates for the PCR products that you
generated and kanamycin plates for the positive and negative control. Plate 2
different volumes to ensure that at least one plate will have well spaced colonies.
An efficient TOPO cloning reaction should produce many colonies. Most colonies
should be white.
18. Pick the white colonies for analysis as these should have the insert. The dark blue
colonies have -galactosidase activity and these lack the insert.
19. Grow several white colonies overnight in 5 ml of LB medium containing 50
g/ml ampicillin or kanamycin.
20. Prepare a glycerol stock for long term storage. Streak the original colony out on
an LB agar plate containing 50 g/ml of ampicillin. Isolate a single colony and
inoculate into 1-2 ml of LB medium with antibiotic and grow overnight. Mix 0.5
ml of culture medium with 0.5 ml of glycerol solution and add to a cryovial. Place
in the -70ºC freezer.