Download Exercise 5. DNA Ligation, Selection and

ChE 170L Laboratory
Exercise 5. DNA Ligation, Selection and
Screening of Ligation Products
Objectives
The techniques learned in Exercises 2 – 4 will be applied in this experiment in order to
complete a subcloning exercise. A piece of DNA that has been cut with restriction
endonucleases will be mixed with a plasmid vector that has been cut with complementary
enzymes. The two pieces will be covalently linked through the action of DNA ligase, forming a
new plasmid. Competent E. coli cells will be transformed with the ligation reaction. Individual
colonies will then be chosen and screened to identify the ligation products.
PreLab Questions 5
Please limit your answers to a few sentences IN YOUR LAB NOTEBOOK
1. Using what you know of ligation, what factors may be important when choosing a vector
for the expression of your gene of interest?
2. Why are multi-cloning sites preferred locations for the insertion of a gene?
3. What role does ATP play in the DNA ligase reaction (be specific)? How might the lack
of a 5’ phosphate group affect this mechanism?
4. Is it possible for a vector that has failed to incorporate the gene during ligation to instead
stick to an identical vector resulting in a cDNA twice the size expected? Why or why
not? Would this be more common with sticky ends or blunt ends?
Background
The advent of recombinant DNA technology is arguably the most important advancement
in applied biology to be developed last century. One aspect of recombinant DNA technology
allows different segments of DNA to be assembled through a “cut and paste” procedure. In this
case, the molecular “scissors” are restriction endonucleases. The “glue” is the DNA ligase
enzyme. When two pieces of DNA that have been cut with EcoRI, for example, are mixed
together in a buffered solution, diffusion through the liquid will eventually cause the compatible
“sticky” ends of the DNA to come together. The hydrogen bonds between complementary base
pairs will keep the two ends in close proximity. This enables the ligase to seal each of the two
strands of the DNA with a covalent phosphodiester bond, creating a new DNA molecule. In the
figure below, the ligase creates the bonds indicated by the dashes between G-a and A-g.
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ChE 170L Laboratory
...G
...C T T A A
VECTOR
+
+
a a t t c...
g...
insert

...G-a a t t c...
...C T T A A-g...

New Fragment
This very powerful tool allows molecular biologists (and biochemical engineers!) to
introduce a gene like human insulin onto a vector like a plasmid. The plasmid can then be
introduced into a single-celled organism such as E. coli, and the cellular machinery of the host
will produce the corresponding protein. The process by which (1) a gene is identified, (2) a
DNA fragment is obtained containing the gene sequence, and (3) the gene is introduced into a
new host is called cloning. Subcloning occurs when a gene which has already been cloned is
transferred from one vector to another and introduced into a host organism.
pUC19 is one of many plasmids which have been developed specifically for use as
cloning vectors. (In this instance, cloning also refers to subcloning.) This plasmid carries a
portion of the amino-terminal, or “front,” end of the E. coli gene that produces the enzyme galactosidase. The middle of the coding region of the gene fragment has a stretch of DNA about
50 bp long which contains 11 unique recognition sequences. An enzyme which recognizes one
of these sequences will only cut at this site on the plasmid. The sequences within this stretch of
DNA are collectively called multi-cloning or polycloning sites because they allow several
different enzymes to be used to clone into the same short region of the plasmid. The multicloning sites are not naturally part of the -galactosidase gene coding region, however, they are
in frame. Thus, when the corresponding polypeptide is produced, some extra amino acids are
incorporated but they do not interfere with the normal function of the protein. A host cell must
also be chosen for the transformation step that produces the carboxy-terminal, or “back,” portion
of the gene. These two protein fragments can then come together in the cell to form an active
peptide. This phenomenon is called -complementation.
Production of the -galactosidase enzyme is caused by the addition of a chemical called
IPTG (isopropylthio--D-galactoside). The enzyme can then cleave the chemical X-gal (5bromo-4-chloro-3-indolyl--D-galactoside), producing a blue color. Cells which contain
plasmids that produce -galactosidase are therefore easily identified because they will appear
blue when plated on agar which contains IPTG and X-gal. However, if a foreign DNA segment
is inserted into one of the multi-cloning sites of pUC19, the coding region of the gene for galactosidase will be interrupted and the enzyme will not be produced. In this case, the colonies
will be white (colorless). pUC19 is therefore said to enable blue/white selection because cells
containing plasmids which contain the insert can be selected based on their white color.
In addition to pUC19, which is a cloning vector, you will be using the expression vector
pBAD. This plasmid contains a bacterial promoter to drive high levels of expression in E. Coli
of genes inserted in front of this promoter. Such recombinant protein production was a
revolutionary advance made possible by recombinant DNA technology.
Once competent cells have been transformed with new plasmids and recombinant clones
have been selected, they must be screened to properly identify the plasmid. In the screening
process, individual colonies are chosen and cultured in liquid medium. It is crucial that single,
round colonies be obtained in this step because these represent the descendents of a single cell,
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ChE 170L Laboratory
and that one cell was presumably transformed with a single, unique plasmid. Once the colony of
cells is cultured in liquid medium, the plasmid DNA is isolated and restriction endonucleases can
be used once again to determine (1) whether the insert was successfully ligated with the
linearized vector and (2) the orientation of the DNA insertion.
PROCEDURE
NOTES:
 This procedure will require two laboratory periods to complete. You may choose to write the
entire protocol prior to Day One, or you may write only the portion of the procedure which is
required for a single day prior to that lab period.

DNA ligase, like restriction enzymes, is very expensive and extremely fragile. Do not
contaminate the enzyme, and do not use pipette tips used with enzymes more than once.
Enzymes are generally stored at –20 °C. During this procedure, the ligase should not be held
out of the ice for longer than a few seconds.
Before the lab period, the instructors digested your GFP with EcoR I and Hind III. In addition,
both pBAD and pUC19 were digested with EcoRI and Hind III. In this lab, they will be
electrophoresed and purified from the gel for use in the ligation reaction.
Gel Extraction and Ligation (Day One)
You will be cutting out GFP, pBAD and pUC19. Note that pBAD and GFP will be in the same
lane.
1. Excise the DNA fragment from the agarose gel with a clean, sharp razor
2. Weigh the gel slice in a microcentrifuge tube. Add 100uL of buffer QG for every 100mg of
gel slice (1 gel volume)
3. Incubate this mixture at 50C for 10 minutes, taking out to shake at 5 minutes. This will
dissolve the agarose gel and release the DNA.
4. After gel slice is dissolved completely, add 1 gel volume of isopropyl alcohol and mix.
5. Apply sample to the spin column and centrifuge at 13k rpm for 1minute. Max volume is
800uL, if you have more than this, add 800uL, spin, then add the rest and spin again. This
binds the DNA to the column
6. Add 0.5mL of buffer QG to the column and centrifuge for 1 minute. This dissolves and
discards any leftover agarose
7. Add 0.75mL of buffer PE to the column and centrifuge for 1 minute.
8. Discard the flow through and centrifuge the column again for 1 minute
9. Place column in clean 1.5mL centrifuge tube.
10. Add 30uL of buffer EB to the center of the column and let sit for 1 minute. Then centrifuge
for 1 minute. This 30uL contains the purified digested fragment.
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ChE 170L Laboratory
11. LIGATION BEGINS HERE. Set up the following ligation reactions in 1.5 ml microfuge
tubes:
Reaction 1:
6 l water
4 l digested pBAD vector
7 l digested GFP
2 l T4 DNA ligase buffer
1 l T4 DNA ligase
Reaction 2:
6 l water
4 l digested pUC19 vector
7 l digested GFP
2 l T4 DNA ligase buffer
1 l T4 DNA ligase
12. Mix the reactions by pipeting up and down gently. Incubate overnight at 14oC.
Transformation (Day Two)
13. Transform the ligation reactions and a control plasmid into competent bacteria. Use 10 l of
the ligation reaction and 5 l of the plasmid control in the transformation.
Remove four tubes with 100 l competent cells from the dry ice cooler and thaw on wet ice.
Label the tubes to match your ligation reactions or control plasmids.
The control plasmid is pUC19 plasmid DNA (obtained from GSI).
Spread cells onto LB-AMP-IPTG-Xgal plates.
Place plates in the 37°C incubator for overnight culture.
Observation of Cultures
14. Place your pBAD-GFP plates on the UV transilluminator. Observe the colors of the colonies
present under UV light.
15. Observe the pUC19-GFP plates by room lighting, not UV.
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ChE 170L Laboratory
Guidelines for Analysis & Conclusions Section
(Remember, these are points you should consider and include in your analysis. This section,
however, need not be limited to these specific guidelines.)
1. Why is it necessary to run the digestion mixture on a gel prior to ligation? Is it necessary to
ligate your products immediately after extracting them from the gel? Why or why not?
2. Discuss why the pBAD-GFP bacterial colonies are green on LB-AMP-IPTG-Xgal plates.
Discuss why the pUC19-GFP colonies are white and not green or blue.
3. pBR322 was one of the first plasmids designed for use as a cloning vector. This plasmid
contains genes for resistance to two antibiotics, ampicllin and tetracycline. There are a few
unique restriction sites in the coding regions of each antibiotic resistance gene. Describe a
method for selection of recombinant plasmids which have been produced from pBR322.
4. The restriction enzyme AvaI has the following recognition sequence: C Py C G Pu G. “Py”
indicates a pyrimidine (C or T), and “Pu” indicates a purine (A or G). How many AvaI
restriction sites would you expect to observe in a 4.2 kb plasmid?
EQUIPMENT AND REAGENTS
pUC19 and pQE40 plasmid vector DNA cut with Bam HI and Hind III
GFP PCR insert fragment cut with Bgl II and Hind III
Sterile nanopure water
T4 DNA ligase, supplied with 10X reaction buffer from New England Biolabs
XL-10 Gold® competent cells
Sterile LB medium, with and without 100 g/ml ampicillin
Six LB agar plates with 100 g/ml ampicillin, 32 g/ml IPTG, 32 g/ml X-gal
Disposable cell spreaders
Alcohol lamp
Ice bucket w/ice
37°C and 42°C water baths
0.5 ml microcentrifuge tubes, sterile and non-sterile
1.5 ml microcentrifuge tubes, non-sterile
Sterile micropipet tips
Promega Wizard Miniprep DNA Isolation Kit
Restriction Enzymes: Nco I, EcoRI and Hind III, from Roche Biochemicals and New England
Biolabs, respectively, supplied with 10X reaction buffers
Electrophoresis unit. Includes power supply, box, plate, and sample comb.
Gel camera
Polaroid Film for gel pictures
UV light box
Commercial Molecular Weight Marker, 1 kb DNA ladder from New England Biolabs
Ethidium Bromide (EtBr): Stock Solution at 10 mg/ml
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ChE 170L Laboratory
Agarose (Electrophoresis-grade)
10X loading buffer (see Ex 3 for composition)
50X TAE Gel Electrophoresis Buffer (see Ex 3 for composition)
REFERENCES
Bailey, J.E., and Ollis, D.F. (1986) Biochemical Engineering Fundamentals, 2nd Ed., pp. 340344, McGraw Hill, Inc., New York.
Blanch, H.W., and Clark, D.S. (1996) Biochemical Engineering, pp. 533-538, Marcel Dekker,
Inc., New York.
Sambrook, Fritsch, and Maniatis. (1989) Molecular Cloning - A Laboratory Manual, 2nd ed., pp.
1.85 – 1.86, Cold Spring Harbor Laboratory Press.
Voet, D., Voet, J.G. (1990) Biochemistry, pp. 824-829, John Wiley and Sons, New York.
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