LABORATORY 2: ELECTROPHORESIS, 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.
In general, for ligation of DNA fragments into a plasmid, the ligation mixture should
include a 3:1 molar ratio of DNA fragment: plasmid DNA.
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.
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.

Make 2 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. You
should come in sometime Sunday afternoon to select colonies and make 3ml
cultures to use on Monday.
Exercise 3. Gel Electrophoresis of gDNA
The standard method for separating DNA fragments is electrophoresis through
agarose gels. DNA is applied to a slab of gelled agarose and then an electric current is
applied across the gel. Because DNA is negatively charged (phosphate groups in the
backbone), it migrates through the gel towards the positive electrode. The rate of
migration depends on:
1) the size of the fragment; the smaller the DNA, the faster it "runs" through the gel;
2) the concentration of agarose; the higher the concentration, the more the agarose retards
the movement of the DNA fragments; and
3) the voltage applied to the gel; the higher the voltage the quicker the DNA "runs" (but
at a trade-off--the DNA fragments do not separate as efficiently).
a. Pouring the gel
A demonstration on preparing and running an agarose gel will be given. Each group
should have a gel apparatus and cover, two end walls, two combs and a plastic gel tray.
The gel tray should be placed in the chamber and the two end walls inserted. A comb
should be placed near one of the end walls.
Make 100 ml of a 1.0% agarose in 1X TBE for your gel in a 250 ml flask. Minigels
require ~30 ml agarose.
Use the microwave to melt the agarose. Be sure the solution is completely melted and
homogenous.
Warning!! Never leave the agarose solution unattended when using the microwave. The
solution must be frequently swirled during the heating process to prevent superheating of
local areas. Always use "hot hands" or autoclave gloves when heating the agarose.
You will be provided Ethidium Bromide (EtBr) at a concentration of 1 mg/ml in a 1.5
microfuge tube. This tube should be saved and stored at 4ºC.
Warning!! Ethidium bromide is a mutagen. Remember to wear gloves when working
with EtBr and to dispose of contaminated tips in specially marked containers. Gels
should be placed in a plastic bag for disposal. Microwaving EtBr produces harmful
volatiles.
Add EtBr to a final concentration of 0.1 to 0.5 g/ml to agarose immediately before you
pour the gel. To do this, label a 50 ml conical tube EtBr/agarose. Add the EtBr to the
tube and then pour about 30 mls of the agarose into the tube. Cap and invert a few
times. The agarose solution should be cooled to about 50ºC (a temperature at which one
can hold the flask, but it is uncomfortable) before pouring the gel. Pouring the gel when
the agarose is too hot may damage the gel apparatus.
Pour the 1.0% agarose minigel with 0.1 g/ml EtBr and allow it to solidify.
Practice loading a few lanes with loading dye if you've never loaded a gel before. This
can be run into the gel without affecting the migration of DNA added to the well at a later
time.
At first it will be easier to load the wells dry. Add 1X TBE buffer to submerge the gel
prior to electrophoresis. Later, you should become adept at loading wells submerged in
buffer.
b. Loading the Samples and Running the Gel:
To a new eppendorf or on parafilm, add 5 l loading dye to 15 l of your sample and
finger flick. Load 20 l total volume of your samples per well for minigels with 8-well
combs. Load 10 l of the molecular weight markers provided. Your gel should contain:
1. molecular weight markers
2. gDNA group 1
3. gDNA group 2
4. gDNA group 3
Run the gel at 100 V until the bromophenol blue is about 3/4 through the gel (approx. 1 h).
Using gloves, carefully remove the tray with the agarose gel. Take it to the UV
transilluminator and slide the gel off the tray onto the lamp. Wearing UV-protective
goggles (NOT YOUR SAFETY GLASSES), examine the gel. The cover of the
transilluminator may also be used to protect your eyes. Photograph your gel with a ruler
for your notebook.
Conclusions.
What did your digests tell you?
Did you get the desired insertion?