LABORATORY 2: LIGATION AND BACTERIAL TRANSFORMATION
The TAs will amplify a gene of interest using PCR and supply you with the amplified
DNA for ligation.
Exercise 1. Ligation of the PCR product into the TOPO vector.
For ligation, we will use the TOPO TA cloning kit by Invitrogen. It allows for efficient
insertion of PCR products into a plasmid vector in a single step, at room
temperature, in 5 min. It relies on the phenomenon that Taq polymerase has a
nontemplate-dependent terminal transferase activity that adds a single
deoxyadenosine (A) to the 3’ ends of PCR products. The linearized vector
supplied with the kit has single, overhanging 3’ deoxythymidine (T) residues that
allow PCR inserts to ligate efficiently with the vector.
See attached protocol
Be sure to include a vector only control: a ligation reaction with no fragment DNA.
This will give you an idea of the background ligation (the vector religating
without insert). This could be prevented by dephosphorylation of the linearized
vector but doesn't always work completely.
Ligation of DNA in a mixture routinely results in a variety of potential products.
1. Only circularized plasmids will survive in the bacteria. This eliminates all
ligation products that are not recircularized.
2. In addition to our gene of interest, the vector plasmid contains an ampicillin
resistance gene. We will grow the bacteria on nutrient agar containing
ampicillin. This will eliminate extraneous bacteria as well as any circularized
products without the appropriate antibiotic resistance.
3. Blue/white selection (Lac selection) – in addition (or instead of)
dephosphorylation to eliminate religation of the vector, blue/white screening
can be used. The multi cloning site in the plasmid interrupts a lacZ’ gene.
lacZ’ codes for part of the enzyme -galactosidase. X-gal (5-bromo-4-chloro3-indolyl--D-galactopyranoside), a lactose analogue, is broken down by galactosidase to produce a product that is deep blue. Thus, if ligation is
successful, lacZ’ is interrupted, a competent -galactosidase is not formed,
and the colony is white. If ligation fails, lacZ’ is translated, a competent galactosidase is formed, and the colony is blue.
Once ligation is completed, transformation of E. coli will allow for amplification of the
product (or products). After the ligation products have been amplified (so we have
something to work with), we could do a plasmid DNA extraction and a series of
restriction digests to determine if we accomplished our goal of inserting the gene of
interest into the vector. These digests are routinely referred to as diagnostic digests.
Unfortunately, we will not have time to do this.
Exercise 2. Bacterial transformation
Why and how bacterial cells can be made to be competent for DNA uptake is not
understood. Fortunately, they can be made competent, and they can be stored in
this state indefinitely at -80C.
Bacteria can be transformed in a variety of ways. Some cells are chemically competent
and required only the appropriate chemical to be transformed. Others are
competent for electroporation and require an electrical shock to create pores in the
membrane. Still others are competent for transformation by heat shock.
We will use heat shock for transformation of our ligation product. The DNA does not
need to be purified at this stage. The ligation mixture will not affect
transformation by heat shock.
Depending on the cells and the source of the cells, techniques for transformation may
vary slightly. Always follow the instructions on the package insert to perform
your transformation.
Protocol:
See attached protocol
The last step of the transformation procedure is to plate the mixture onto nutrient plates
with antibiotic selection (We will use ampicillin.) and incubate overnight at 37C.

You will be provided two types of plates: 1) those withX-gal (5-bromo-4chloro-3-indolyl--galactopyranoside) and IPTG (isopropylthiogalactoside), an inducer of the enzyme, for blue/white selection and 2)
those without X-gal/IPTG for the Southern analysis.

Make 4 plates: 40 l of your transformation solution onto each plate
80 l of your transformation solution onto each plate
Colonies should appear by the next day. The TAs will put move your plates to 4oC until
Monday when we do the colony lift.
Exercise 3. Beginning Southern Analysis of a Gel
In 1975, E. M. Southern developed a technique to transfer DNA directly from an agarose
gel to a nitrocellulose or nylon membrane. The technique relies on capillary action. The DNA,
once bound on the membrane, can then be used for hybridization analysis to determine the
presence and location of a particular DNA sequence. In the past decade, Southern's original
paper on the technique was the most frequently cited paper in research.
Materials needed:
0.2 N HCl
DIG High Prime labeling and detection kit
0.5 M NaOH/1.5 M NaCl
20X, 2X SSC
1.5 M NaCl/0.5 M Tris-HCl, pH 7.4 0.2M EDTA (pH 8.0)
Nitrocellulose membranes
paper towels
Whatman 3MM filter paper
glass plate
sponge
Pyrex dish
weight
Tupperware dish/or sealable bag
Shaking incubator or water bath, temperature controlled
Setting up the gel for transfer
Wash the gel in: 10 min in 0.2N HCl
water several times
15 min (twice) in denaturation solution (0.5 M NaOH/1.5 M NaCl)
30 min in neutralization solution (1.5 M NaCl/0.5 M Tris-HCl, pH 7.4)
Prepare the transfer setup: Place a sponge in the bottom of a glass tray and fill the dish half
way with 20X SSC. When the sponge is fully saturated, layer 3 pieces of Whatman 3MM paper.
Wet the filter paper thoroughly and remove all air bubbles. Drain the gel and lay on top of the
filter paper being careful to avoid air bubbles. (Clip off one corner of your nitrocellulose and
record which corner you removed. This will help you orient your blot to the picture of your gel.)
Lay the nitrocellulose exactly on top of the gel. Be sure the nitrocellulose does not overhang the
gel and remove any trapped air bubbles. Now add, in order, 3 more pieces of Whatman filter
paper, 2" of paper towels, a glass plate and top it off with a small weight. Cover the edges of the
tray with plastic wrap. Allow the transfer to proceed overnight.