Genome Sequence Databases: Genomic, Construction of Libraries
J M Struble, P Handke, and R T Gill, University of Colorado, Boulder, CO, USA
ª 2009 Elsevier Inc. All rights reserved.
Defining Statement
Vectors Used in Genomic Library Construction
Genomic DNA Preparation
Agarose Gel Electrophoresis Used in Genomic Library
Construction
Quantifying DNA and Determining Quality
Ligation Reactions
Transformation of Library DNA into Bacterial Host
Strains
Glossary
agarose gel electrophoresis Movement of charged
molecules, such as proteins and nucleic acids, induced
by an electrical field.
blunt ends DNA fragment ends with no overhanging
nucleotides resulting from enzyme digestions.
cohesive ends DNA fragment ends with
single-stranded overhangs resulting from enzyme
digestions; also known as sticky ends.
competent cell Cells treated to accept extracellular
DNA.
copy number Number of particular plasmids present in
a cell.
electroporation The process of introducing vectors
into a host organism by temporarily creating
electropores to allow DNA passage.
end repair Process employing Klenow fragments or
DNA polymerases to fill in or remove overhangs
obtained by restriction enzyme digestions.
genomic library Collection of overlapping segments of
DNA including all regions of an organism’s genome.
ligase Enzyme catalyzing the reformation of
phosphodiester bonds to join two compatible DNA
fragments.
Abbreviations
CFU colony forming units
Defining Statement
Genomic libraries are becoming more important as the uses
for biotechnology multiply and expand at an increasing
Increasing the Number of Transformants
Determining the Number of Transformants Needed for
Coverage of an Entire Genome
Current Strategies to Enhance Genomic Library
Production
Sample Protocol to Construct a 4 kb Pseudomonas
aeruginosa PAO1 Library with Vector PBTB-1 Within
an E. coli Host Strain
Further Reading
ligation Joining of two compatible fragments of DNA
through the reformation of phosphodiester bonds
catalyzed by ligase.
partial digestion Enzyme digestion performed for a
limited amount of time that does not go to completion
for purposes of generating random DNA fragments for
library construction.
plasmid Extrachromosomal, circular DNA molecule
employed to introduce DNA fragments into a host
organism.
recombinant clone Clone containing a recombinant
DNA molecule.
restriction enzyme Enzyme recognizing a specific
DNA sequence around which it will cleave both strands
of a DNA segment.
transformation Introduction of foreign DNA into a host
organism.
vector DNA molecule, such as a plasmid, into which
exogenous DNA fragments can be ligated for
transformation into a host organism and propagation
within that host.
LB broth Luria–Bertani broth
pace. Identifying and isolating genes of interest, as well as
gene mapping and sequencing, must be preceded by con-
struction of a genomic library. The quality and specific
features of such a library are therefore of utmost importance.
185
186 Techniques | Genome Sequence Databases: Genomic, Construction of Libraries
Vectors Used in Genomic Library
Construction
Vector Selection
The choice of backbone vector used for constructing a
genomic library is highly dependent upon what studies
will be performed. When choosing a backbone vector,
decisions must be made about the desired copy number,
selective marker, size of genomic DNA insert, host
range, and, if the cloned DNA is to be expressed, the
type of promoter and ribosomal binding site that should
be upstream of the multiple cloning site. Also, the vector
used for library construction should remain stable after
genomic DNA fragments are inserted.
For constructing a genomic library, it is important that
the vector has the ability to replicate within the desired
host organism or organisms. The origin of replication, or
the ori region, on a vector controls the host range and, to a
large extent, the copy number of the vector. Vectors with
narrow host ranges such as those containing the ColE1
origin have only been found to replicate in Escherichia coli
and closely related bacteria. Broad host range vectors have
origins of replication that are recognized in a wide range of
bacterial species. The origin of replication from the broad
host range plasmids RK2 and pBBR1 is functional across
multiple Gram-negative species while plasmid RSF1010
has been found to be able to replicate within a number of
both Gram-positive and Gram-negative species.
The ori region (replicon) of a vector also affects the copy
number of vector within the host. High copy number vec-
tors offer the advantage of increased yield of purified vector
from a volume of culture to be used for sequencing or
molecular cloning purposes. Low copy number vectors
are typically desired for studies involving expression of
genomic DNA segments, especially those involving toxic
DNA segments. When a higher yield of a low copy number
vector is needed, low copy number vectors containing the
ColE1 or pMB1 origin of replication, which allows for
plasmid number amplification prior to purification in the
presence of 170 mg l1 of chloramphenicol, may be used.
Chloramphenicol inhibits protein synthesis and thus pre-
vents chromosomal replication. The enzymes necessary for
replication of plasmids with the ColE1 or pMB1 origin
require enzymes that are long lived and thus continue to
replicate in the presence of chloramphenicol, reaching sev-
eral thousand copies per cell.
The ori region and the mechanism of vector replication
have also been implicated to be responsible for the structural
stability of cloned vectors. Vectors with replicons using roll-
ing circle mechanisms have frequently been found to be
unstable for cloning purposes. This instability may be due
to the secondary structure formed by the lagging strand of
DNA during replication, which may be cleaved by nucleases
or experience mutations or deletions during replication.
Features of and around the insert site also have an
impact on stability and representation of the library. For
the creation of a representative genomic library, all DNA
sections of the genome must be cloned, regardless of
DNA secondary structure or the encoding of toxic gene
products or strong promoters. Vectors that contain tran-
scription terminators flank the cloning site to prevent
transcription into and out of the cloned DNA and have
been shown to be successful at mitigating cloning bias
against difficult DNA and increase vector stability. For
studies involving expression of the insert DNA, a variety
of vectors exist that offer options of using constitutive,
inducible, titratable, or native promoters, as well as
options including ribosomal binding sites and start codons
upstream of the cloned insert.
Vector Preparation
Once an appropriate vector has been selected, it must be
prepared for cloning. Purified vector should be free of
endonuclease contamination and chemicals such as phe-
nol or EDTA that may interfere with downstream
enzymatic reactions. There exist a number of standard
protocols and commercially available kits that are used
for purifying plasmid DNA. Plasmid purification proto-
cols take advantage of the smaller size of the plasmid
DNA compared to the significantly larger chromosomal
DNA. The method chosen is influenced in part by the size
of the plasmid and the host strain. Many commonly used
cloning strains, such as E. coli DH5, have mutations
within their endA regions, which eliminate the nonspecific
endonuclease activity of endonuclease I, thus allowing
higher quality plasmid preparations from these strains.
Other strains such as E. coli HB101 produce large amounts
of carbohydrates that can interfere with DNA extractions.
Large plasmids (>15 kb) are more fragile and thus have to
be treated with more gentle extraction methods than
small plasmids, which are less susceptible to damage.
Once purified, a vector needs to be linearized prior to
ligation. Using the sequence of the vector and the vector
map, an enzyme or a set of enzymes must be found that
cut only within the cloning site of the vector. Enzymes
may leave blunt or sticky ends after digestion. When
selecting enzymes to digest the vector, it must be kept
in mind that the ends for the vector and the ends for the
fragmented genomic DNA that will be cloned must be
compatible. The sticky ends of a digested piece of DNA
may be converted to blunt ends by using particular DNA
modifying enzymes that may fill in overhangs of sticky
ends or exhibit exonuclease activity against single-
stranded DNA and thus degrade overhangs. T4 DNA
polymerase or the Klenow fragment of E. coli DNA poly-
merase I will remove 39 overhangs and fill in 59 overhangs
when provided with deoxynucloside triphosphates.
Additionally, DNA end repair kits are available from
 Techniques | Genome Sequence Databases: Genomic, Construction of Libraries
several commercial vendors that use proprietary enzymes
to generate blunt-ended DNA segments.
In an effort to prevent self-ligation and minimize the
number of clones without genomic DNA insert, the
59-termini phosphate groups required by ligase for ligation
reactions are removed from the linearized vector using an
alkaline phosphatase. The most commonly used alkaline
phosphatases within molecular biology are shrimp alkaline
phosphatase from the Arctic shrimp Pandalus borealis, calf
intestinal alkaline phosphatase from calf intestines, bacterial
alkaline phosphatase from E. coli C4, and Antarctic
Phosphatase from the psychrophilic bacterium strain
TAB5. All of these phosphatases are effective at removing
59 phosphates from DNA, but vary in their activity, buffer
compatibility, and ability to be inactivated. Alkaline phos-
phatases bind tightly to DNA and thus may require
aggressive methods to denature. To inactivate bacterial
alkaline phosphatase and calf intestinal alkaline phosphatase
reactions, proteinase K is used to digest the phosphatase,
followed by a phenol–chloroform extraction and an ethanol
precipitation. Alternatively, a commercial enzymatic reac-
tion clean-up kit can be used to purify the
dephosphorylated DNA. Shrimp alkaline phosphatase and
Antarctic Phosphatase from TAB5 are thermolabile and can
be completely heat inactivated. It desired to use an alkaline
phosphatase that is compatible with the restriction enzyme
buffer or buffers that were used in upstream preparation of
the vector and to have an alkaline phosphatase that is heat-
labile to minimize the number of purification steps, which
may decrease the yield of vector DNA.
The dephosphorylation step, which removes 59 phos-
phate groups from the linearized vector, may not go to
completion and thus the amount of background from
vector that can self-ligate may be significantly high. An
optional step to help mitigate this problem is to perform a
ligation reaction following the dephosphorylation step.
Vector DNA that maintained the 59 phosphate groups
will self-ligate while linearized vector DNA that has had
its 59 phosphates successfully removed will be incapable
of self-ligating and thus remain linear. The circular self-
ligated vector DNA and the linear vector DNA can then
be separated by agarose gel electrophoresis. The linear-
ized vector can be extracted with a scalpel and purified
using traditional DNA extraction methods or commer-
cially available kits for DNA extraction from agarose gels.
If the ligation step to remove vectors that had not been
successfully dephosphorylated is omitted, the linearized
vector should still be purified from the restriction enzyme
digestion and the alkaline phosphatase reaction buffers and
enzymes. Phenol–chloroform purification followed by an
ethanol precipitation or a commercially available spin col-
umn for cleanup of enzymatic reactions may be used for
this purpose. Purifying the vector DNA will remove pro-
teins and buffer components that may lessen the efficiency
of the ligation of the vector to genomic DNA insert.
187
PCR amplification may also be used to generate large
amounts of linear dephosphorylated vector. Culture-
purified and linearized vector is PCR amplified with
unphosphorylated primers designed to extend outward
from the cloning site into the vector backbone.
Proofreading polymerases such as Pfu or Pfx DNA poly-
merase will generate high-fidelity blunt-ended PCR
product. The choice of polymerase influences the types of
overhangs that will be generated as well as the fidelity of the
PCR product generated. The PCR product should be pur-
ified away from the components in the PCR buffer prior to
ligation so as not to have extraneous nucleotides, primers,
or salts that may interfere with enzymatic reactions. To do
so, the sample may be separated via agarose gel electro-
phoresis followed by excision of the appropriate band
containing the PCR product of linearized vector. DNA
can then be extracted via a commercial gel extraction kit.
Genomic DNA Preparation
Genomic DNA Purification
Genomic DNA must be isolated from proteins and other
cellular debris prior to any enzymatic or mechanical
manipulation. Bacterial cells are lysed, typically through
exposure to surfactants, such as sodium dodocyl sulfate or
Tween-20, or treatment with lysozyme to digest the
polysaccharide component of cellular membranes and
proteinase K for protease digestion. DNase-free RNase
may be added to the lysis step to minimize RNA contam-
ination. Genomic DNA can be purified from cell lysate
using a phenol–chloroform extraction followed by an
ethanol precipitation or commercially available silica col-
umns. Commercially available kits for genomic DNA
isolation are often desirable in that they avoid the use of
phenol and chloroform, which are toxic and may interfere
with downstream enzymatic reactions. Many commer-
cially available kits avoid phenol by using buffers
containing the chaotropic agent guanidine hydrochloride
to aid in cell lysis and to effectively denature proteins.
Purified genomic DNA should be maintained in a nucle-
ase-free Tris buffer or in nuclease-free water.
Nuclease contamination is a frequent concern asso-
ciated with genomic DNA isolation. Nuclease activity
will degrade DNA and can be easily mistaken for a
restriction enzyme digestion or the result of mechanical
shearing. Nuclease contamination may be detected by
incubating an aliquot of purified DNA at 37  C for 18 h
and then visualizing the DNA on an agarose gel. A control
aliquot of DNA that had been stored frozen should be
used for comparison. Following electrophoresis, if the
incubated aliquot appears to have degraded, nucleases
may be contaminating the genomic DNA sample.
Additionally, the DNaseAlert kit available from Ambion
(Austin, TX) can be used to detect DNase contamination
188 Techniques | Genome Sequence Databases: Genomic, Construction of Libraries

in a sample. This kit detects DNase in a sample through

the use of modified oligonucleotides that fluoresce upon
cleavage by nucleases that may be within the sample. If
nuclease activity is detected, the DNA sample can be
exposed to high concentrations of guanidine hydrochlor-
ide (6–8 M) for 30 min or be subjected to an additional
phenol–chloroform extraction for more thorough depro-
teinization of the sample. An ethanol precipitation should
follow the additional deproteinization step to remove the
phenol, chloroform, or chaotropic salts.

Fragmentation
Once purified and established as free of contaminating
nucleases, genomic DNA must be fragmented to a desired
size and made compatible for ligation into a prepared
vector. Genomic DNA may be fragmented using enzy-
matic or mechanical means. Enzymatic fragmentation is
accomplished using either restriction endonucleases or
DNase I in the presence of manganese ions. Digestion
with DNase I offers the ability to generate a more random
pool of DNA segments compared to digestion with
restriction endonucleases, which are biased based on the
sequence of the genomic DNA and the recognition sites
of the enzymes. Despite this, DNA digestion with restric-
tion endonucleases is often preferred due to simplicity in
reaction set-up and controllability. Appropriate restric-
tion endonucleases for genomic DNA digestions are
chosen based on five factors:
1. Frequency of cutting.
2. Buffer compatibility.
3. Ability to be denatured. Figure 1 Restriction enzyme digestion of genomic DNA for
4. Methylation sensitivity. 5 min (middle) and 16 h (right) next to DNA standard ladder (left).
5. The type of overhang that is produced.
Restriction endonucleases have known recognition sites enzymes may be chosen based on the type of overhang that is
ranging from 4 to greater than 30 nucleotide bp. Their left after cleavage. It can be arranged to have blunt ends or
frequency of cutting within a genome may be predicted if overhangs (called cohesive ends) that would be compatible
information is known about the genome sequence and the with the prepared vector. This will eliminate a step to
recognition sequence of the enzyme. Enzymes that cut with modify the ends of the DNA fragments prior to ligation.
a high frequency in the genome, typically containing smaller Mechanical methods for DNA fragmenting offer the
recognition sequences, can be used to generate suitably advantage of being unbiased toward DNA sequence and
random fragments by using partial digestions (Figure 1), or thus are useful for creating a more random pool of
digestions that have not gone to completion. More than one fragments. The main disadvantage associated with
restriction enzyme may be used for the partial digestion of mechanically fragmenting DNA is the limitation on the
DNA to ameliorate bias based on recognition sequence. size of fragments that can be generated as well as the
Ideally, all of the restriction enzymes used in a digestion extensive treatment that is required to repair the ends of
should have similar activity within a common reaction buffer the DNA necessary before they can be cloned into back-
and ability to be denatured to minimize DNA purification bone vector. A French press, sonicator, clinical nebulizer,
steps. Additionally, the restriction enzymes selected may be small gauge syringe, and HydroShear (Genomic Solutions
desired to be insensitive to dam methylation (methylation of Inc., Ann Arbor, MI) are common tools used to fragment
the N6 position of the adenine in the sequence GATC) or DNA. Of these tools, the HydroShear was deliberately
dcm methylation (methylation of the cytosine at its C5 designed to shear DNA using hydrodynamic force and
found in the sequences CCTGG and CCAGG), which can be used to reproducibly create random fragments of
may render the DNA resistant to cleavage. Lastly, restriction DNA within a limited size range, independent of DNA
 Techniques | Genome Sequence Databases: Genomic, Construction of Libraries 189

concentration, starting length of DNA, and sample volume. 100%

Mechanical fragmentation of DNA will result in DNA that 10%
must be end-repaired to fill in or remove overhangs and 1%
restore 59 phosphate groups. T4 DNA polymerase, Klenow
0.1%
fragment, or kits available to repair DNA ends may be used
for this purpose. T4 polynucleotide kinase, when supplied 0.01%

with ATP, can be used to restore 59 phosphate groups 0.001%

No UV 30 s 60 s 120 s 120 s
necessary for ligase activity. 302 nm 302 nm 302 nm 360 nm

Figure 2 Relative cloning efficiency of pUC19 after exposure to

short or long wavelength UV light. Intact pUC19 DNA was
Agarose Gel Electrophoresis Used transformed after no UV exposure (‘No UV’) or exposure to
in Genomic Library Construction 302 nm UV light for 30, 60, or 120 s (‘30 s 302 nm, 60 s 302 nm,
120 s 302 nm’) or to 360 nm UV light for 120 s (‘120 s 360 nm’).
Cloning efficiencies were calculated relative to nonirradiated
Agarose gel electrophoresis is used to separate DNA by pUC19 DNA. Reprinted with permission from Lucigen
size or topology using an electric field that induces nega- Corporation (www.lucigen.com).
tively charged DNA molecules to migrate to the positive
pole through a porous matrix of agarose. In library con-
struction, it is used in both vector preparation and
isolation of genomic DNA pieces of a particular size.
Circular vector migrates more quickly through an agarose
gel than linearized vector with the same molecular weight
and thus can be separated from vector that was linearized
after digestion with a restriction enzyme. Vector that has
ligated can also be separated from vector that has not been
ligated and remained linear using gel electrophoresis.
After genomic DNA has been fragmented, pools of gen- (a)
erated segments are separated according to their
molecular weight. DNA segments of less than 20 kbp
can be sufficiently separated with a standard 1% agarose
gel. The resolution of smaller pieces of DNA (<1 kbp) can
be enhanced by increasing the percentage of agarose in
the gel to 2%, while lowering the agarose content to 0.8%
can enhance the resolution of larger pieces of DNA. If
larger pieces of DNA need to be resolved, pulse field gel (b)
electrophoresis (which uses an alternating electric field)
may be employed to achieve higher resolution.
Larger segments or fragile pieces of DNA that are
sensitive to shearing should be resolved using gels made
from low melting point agarose, which does not require
vortexing or centrifugation steps to extract embedded
DNA, but rather relies upon melting the gel at low
temperatures followed by an incubation with -agarase I (c)
to digest the agarose away from embedded DNA.
DNA within an agarose gel must be bound to a dye in
order for visualization. The most commonly used dye to
stain agarose gels is ethidium bromide (EtBr). EtBr is a
fluorescent dye that intercalates between stacked bases of
DNA. EtBr experiences excitation by UV radiation and
emits energy at 590 nm, fluorescing with a red-orange Figure 3 Digested DNA to be used in subsequent steps should
color. Dye bound to DNA displays an almost 20-fold not be exposed to UV light. After loading ladder in lane 1, a
increase in fluorescent yield, thus allowing for the detec- fraction of the digested DNA should be placed in lane 2 with the
tion of as little as 10 ng of DNA. If EtBr is used to visualize rest in lane 3. After running the gel (a), lanes 1 and 2 should be
separated from lane 3 with a scalpel (b) and examined under UV
DNA loaded onto an agarose gel, it is very important to light. The desired DNA fragment should be excised from lane 2,
minimize the amount of short wavelength UV light and both gel pieces should be brought together (c) to obtain the
exposed to the DNA (Figures 2 and 3). Exposure to desired DNA fragment piece.
190 Techniques | Genome Sequence Databases: Genomic, Construction of Libraries
short wavelength UV light has been shown to damage
DNA and decrease its cloning efficiency. In addition,
EtBr is a potent mutagen and moderately toxic, so safer
alternatives or dyes that can be seen without the aid of
UV light are often desired. Alternative dyes include crys-
tal violet, methylene blue, SYBR Safe(Invitrogen,
Carlsbad, CA), or Nile blue.
Quantifying DNA and Determining Quality
It is important to be able to determine the amount and
purity of DNA within a sample. DNA should be quanti-
fied following purifications steps to be sure of a sufficient
yield prior to any further manipulations. The quality of
DNA should also be monitored before initiating cloning
steps to ensure that there are minimal contaminants that
would interfere with the efficiency of cloning reactions.
The purity of a DNA sample can be accessed by
calculating the ratio of absorbance at 260 nm to the absor-
bance at 280 nm measured by a spectrophotometer.
Nucleic acids have a higher absorbance at 260 nm than
at 280 nm. The reverse is true for proteins, which display
higher absorbance at 280 nm than at 260 nm. The absor-
bance at each individual wavelength is thus influenced by
the presence of both proteins and nucleic acids. Based on
the extinction coefficients for both of these macromole-
cules, pure samples of DNA would have a A260/A280 ratio
of close to 2.0 and pure protein samples would have a
A260/A280 ratio of close to 0.6. Typically, a DNA sample
with a A260/A280 ratio of greater than 1.7 is acceptable for
molecular cloning reactions.
The quantity of DNA can be measured via UV spec-
troscopy, fluorometry, or by comparison to a standard
mass ladder on an agarose gel. Due to the simplicity, the
concentration of DNA within a sample is frequently
approximated based on the absorbance reading at
260 nm. The concentration is found through application
of the Beer–Lambert law, which relates absorbance with
concentration through the relationship
A ¼ "bC
where A ¼ absorbance, b ¼ pathlength of the sample cuv-
ette, in units of length, " ¼ absorption coefficient in units
of volume/mass/length, and C ¼ concentration in units of
mass per volume.
Using a standard spectrophotometer with a path length
of 1 cm, an absorbance reading at 260 nm (A260) of 1
equates to a concentration of approximately 1 ng ml1.
As mentioned above, other molecules besides DNA that
absorb at this wavelength, including proteins, RNA, and
salts within the sample, can influence absorbance at
260 nm. Due to this phenomenon, the amount of
DNA within a sample is usually confirmed or measured
with a different method. Fluorometric measurements of
DNA are more accurate than those obtained from UV
spectroscopy and can detect smaller quantities of DNA.
To quantify DNA, DNA-specific fluorescent stains, such
as PicoGreen or SYBR Green I, are added to a DNA
sample and the fluorescence of the sample is compared to
the fluorescence of standards of known concentrations.
Accurate DNA quantification can also be achieved by
running an aliquot of sample along with a standard
DNA mass ladder on an agarose gel and comparing
DNA band intensities. This method is effective when
quantifying distinctly sized pieces of DNA.
Ligation Reactions
A ligation reaction is required to bind fragmented geno-
mic DNA into linearized vector. The most commonly
used ligase, T4 ligase, is derived from the T4 bacterioph-
age and requires ATP as a cofactor and an available DNA
59 phosphate group on at least one of the two ligating
DNA fragments. When setting up a ligation reaction, the
moles of insert to moles of vector ratio may be varied to
find optimal conditions. Lower ratios may result in ineffi-
cient ligation reactions while higher ratios increase the
risk of ligating more than one insert per vector. Typically,
insert to vector ratios are varied from 1:1 to 5:1. Blunt-
ended ligations may perform best with higher ratios. A
control ligation containing vector without insert should
also be conducted to give an estimate of background
clones that contain self-ligated vector. Ligases may or
may not be required to be inactivated or purified from a
reaction prior to transformation. Following the recom-
mendations of the supplier of the ligase generally will
give the best ligation and transformation results.
Transformation of Library DNA into
Bacterial Host Strains
Naked DNA in solution can be transferred into a bacterial
host strain via transformation of competent cells. Bacterial
transformations with plasmid DNA is accomplished
through heat shock of chemically competent cells or elec-
troporation of electrocompetent cells. Transformation of
chemically competent cells usually achieve 105–109
colony forming units (CFU) per mg of supercoiled DNA
while electroporation of electrocompetent cells can yield
up to 1010 CFU mg1 of DNA.
Generally, the preparation of chemically competent
cultures of E. coli involves treating exponentially growing
cells with a salt solution, such as 0.1 M CaCl2. Plasmid
DNA is mixed with the cells and the plasmid DNA and
cell suspension are heat shocked at 42  C for a brief
period, during which the cells can uptake the DNA.
 Techniques | Genome Sequence Databases: Genomic, Construction of Libraries
While the exact mechanism of DNA uptake by this
method is not fully known, it is believed that the swelling
of the cells following treatment with the salt solution and
the activation of heat shock genes are important in cells
taking up DNA from their environment. Factors that
influence the frequency of transformation include the
purity of the reagents and water used, the viable cell
density of the culture, and the trace contaminants that
are found on glassware. Additionally, the number of times
a culture has been passaged influences transformation
efficiencies. Best results are obtained from cultures started
directly from cryogenic freezer stock as opposed to cells
that have been continuously passaged.
Electrocompetent cells are prepared by repeated
washing of cells in low conductivity solutions such as
10% glycerol or 300 mmol l1 sucrose to reduce the
ionic strength of the cell suspension. Electroporation
works by using a transmembrane electric field pulse to
create small holes, referred to as electropores, within the
bacterial membrane through which DNA can pass.
Electroporation conditions, such as pulse amplitude and
duration, must be sufficient enough to generate electro-
pores but not increased to the point at which the number
and size of electropores detrimentally affect transforma-
tion efficiency by causing cell damage or death. The
number of pulses, along with the pulse duration and
amplitude, can be varied to empirically optimize condi-
tions for various cell lines.
While many bacterial strains can be made competent,
the protocols for preparing and manipulating competent E.
coli are the most thoroughly worked out. Furthermore,
competent E. coli can be obtained from commercial sources.
Commercially available competent cells tend to yield
transformation efficiencies several orders of magnitude
greater than those typically achieved by standard labora-
tory preparations. Additionally, ligated DNA tends to have
lower transformation efficiencies than supercoiled DNA,
Select vector
Purify vector
Linearize vector
End repair vector if necessary
Dephosphorylate vector
Remove residual enzymes and buffers
Ligate vector and genomic DNA fragments
Transform ligation product into bacterial strain
Figure 4 Summary of steps necessary for constructing representative genomic libraries.
191
most likely due to DNA topology. For a combination of
these reasons, the initial transformation step of transferring
cloned library DNA to a host strain is usually conducted
with E. coli, provided that the cloning vector used has an
origin of replication that can be recognized by E. coli DNA
polymerases. If desired, after this initial transformation
step, the extracted supercoiled plasmid DNA can be pre-
pared from transformed E. coli and then transformed into a
different desired host cell line (Figure 4).
Increasing the Number of Transformants
When a large number of recombinant clones is required,
the ligation reaction can be precipitated in the presence of
yeast tRNA. Precipitation of ligation reactions prior to
electroporation has been shown to give up to a 400-fold
increase on the number of transformants. It is believed
that the yeast tRNA alters or stabilizes the topology of the
ligated DNA, increasing its efficiency of transformation.
In this method, a 5 ml ligation reaction is mixed with 1 mg
of yeast tRNA from a 1 mg ml1 solution, brought up to a
total volume of 20 ml with ultrapure water, and then
precipitated with twice the volume of cold absolute etha-
nol. The DNA is pelleted by centrifugation, washed twice
with cold 70% ethanol, and allowed to air dry prior to
resuspension in 1 ml of ultrapure water. This sample can
then be transformed into competent cells.
Determining the Number
of Transformants Needed for Coverage
of an Entire Genome
The extent to which a library represents all sections of the
genome can be statistically determined. The number of
necessary transformants to have sufficient coverage or
Purify genomic DNA
Fragment genomic DNA
End repair genomic DNA if necessary
Run DNA on gel and obtain desired fragment size
Determine quantity and purity of DNA
192 Techniques | Genome Sequence Databases: Genomic, Construction of Libraries
high probability of containing any given section of the
genome is dependent upon the genome size and the size
of genomic DNA inserts contained within a library. The
Clarke–Carbon equation, based on the assumption that
recombinant clones are distributed according to a Poisson
distribution across the genome, can be used to determine
the number of transformants needed to have a high prob-
ability of any given unique DNA sequence that would be
present in a genomic library. The Clarke–Carbon equa-
tion can be written as
lnð1 – P Þ
N¼
lnð1 – f Þ
where N ¼ number of recombinant clones required,
P ¼ probability of finding a given unique DNA section,
and f ¼ fraction of the total genome size that is contained
within a single insert of the genomic library, equal to the
size of the insert in bp per size of the genome in bp.
For E. coli, K12 genome with a 4 639 221 bp sequence, a
genomic library containing 12 000 bp inserts would need
2667 transformants to have a probability of 99.9% of the
library containing any given DNA sequence.
While the Clarke–Carbon equation is the most com-
monly used formula for determining the number of
transformants, other equations, such as the Poisson dis-
tribution-based Lander–Waterman equation may also be
used. The number of recombinant clones required may
also be influenced by the application of the genomic
library. Some applications requiring high amounts of
overlap between DNA segments require higher numbers
of recombinant clones while applications that require
only sections of genes to be present, such as some hybri-
dization studies, may require less transformants.
Current Strategies to Enhance Genomic
Library Production
Large-scale sequencing projects, including the sequen-
cing of entire genomes, require the construction of high
quality and highly representational genomic libraries.
These libraries should have minimal sequence bias and
often it is desired to have clones with larger sized inserts
to decrease the number of clones required to be
sequenced. In order for genomic sequencing projects to
be complete, all DNA of the organism must be included
within the genomic library, including difficult to clone
DNA that contain secondary structures, AT- or GC-rich
regions, or DNA encoding strong promoters or toxic
gene products. Additionally, it is desired for all insert
DNA to be stable within cloning vectors so that the
vector can be amplified or be available for other
molecular biology experiments. To this end, a number
of advances have been developed, particularly in the area
of cloning vectors to improve cloning of genomic DNA
fragments.
Linear vectors based on the coliphage N15, available
commercially from Lucigen (Middleton, WI) such as
pJAZZ vectors, have been shown to be stable for larges
DNA segments (up to 30 kb) or DNA with difficult to
clone secondary structure. The stability of these vectors is
believed to be accredited to their lack of supercoiling and
differences in replication compared to standard cloning
vectors. Low copy number vectors, such as the pSMART
or broad host range pRANGER-BTB series of vectors
(available from Lucigen), have features that block tran-
scription into and out of the multiple cloning site by the
presence of transcriptional terminators and the lack of
constitutive promoters. These vectors have been shown
to be several times more stable for cloning random DNA
fragments than pUC vectors thus minimizing cloning
gaps caused by difficult-to-clone DNA. Another recently
developed vector intended to facilitate sequencing,
pLEXX-AK (also available from Lucigen), is designed to
clone two inserts per vector, thus reducing the down-
stream labor involved in processing clones prior to
sequencing.
Additional advances in constructing genomic libraries
come from a reduction in the amount of work required.
Many molecular biology suppliers now offer kits to aid in
genomic library construction. These kits typically contain
pre-processed vector that has already been linearized and
dephosphorylated, along with prepared competent cells,
reducing the amount of user time required to create a
genomic library. Commercially prepared vectors typi-
cally promise much lower background empty vector
than is usually obtained when cloning vectors are pre-
pared locally.
Sample Protocol to Construct a 4 kb
Pseudomonas aeruginosa PAO1 Library
with Vector PBTB-1 Within an E. coli Host
Strain
Supplies and Reagents Needed
All kits and products should be used according to the
recommendations of the suppliers unless otherwise noted.
• Incubator 
set at 37 C.
• Heat block. set at 37 C.
Water bath 
• Milliliter conical tubes.
• Yeast tRNA (1 mg ml in ultrapure water) (Sigma-
• Aldrich, St. Louis, MO). 1
• Ultrapure water (Invitrogen).
• 100% ethanol.
 Techniques | Genome Sequence Databases: Genomic, Construction of Libraries
• 70% ethanol.
• 80dlacZ
M15
E. cloni 10G (F-mcrA
(mrr-hsdRMS-mcrBC)

lacX74 endA1 recA1araD139
(ara,
leu)7697 galU galK rpsL nupG -tonA) (Lucigen) con-
taining plasmid pBTB-1 (-lactamase resistance, low
copy number).
• P. aeruginosa PAO1.
• Luria–Bertani (LB) broth.
• YT agar plates supplemented with 100 mg ml1
carbenicillin.
• Sterile-filtered stock solution of carbenicillin
(100 mg ml1).
• HiSpeed Plasmid Midi kit (Qiagen, Valencia, CA).
• 500/G Genomic-tips (Qiagen).
• Genomic DNA Buffer Set (Qiagen).
• MinElute Gel Extraction Kit (Qiagen).
• QIAprep Spin Miniprep Kit (Qiagen).
• Nuclease-free water (Ambion).
• Agarose gel electrophoresis apparatus.
• 1  TAE buffer (50: 242 g Tris base 57.1 ml acetic
acid 100 ml 0.5 M EDTA. Add deionized water to 1 l
and adjust pH to 8.5) to be used for all electrophoresis
steps and in preparing agarose gels.
• Molecular biology grade agarose (Sigma-Aldrich).
• Low-melting point agarose (Promega, Madison, WI).
• 10 mg ml1 stock solution of EtBr.
• 10% sodium dodecyl sulfate solution (Ambion).
• GELase Agarose Gel-Digesting
(Epicentre Biotechnologies, Madison, WI).
Preparation
• HincII (New England BioLabs, Ipswich, MA).
• RsaI (New England BioLabs).
• HaeIII (New England BioLabs).
• Antarctic Phosphatase (New England BioLabs).
• T4 DNA ligase (Lucigen).
• High DNA Mass Ladder (Invitrogen).
• 1 kb Plus DNA Ladder (Invitrogen).
• UltraClone DNA Ligation & Transformation Kit con-
taining ELITE DUOS (Lucigen) (contains ligase and
E. cloni ELITE 10G electrocompetent cells).
Vector Preparation
1. Inoculate 100 ml of LB broth supplemented with
100 mg l1 carbenicillin and allow to incubate over-
night at 37  C, 225 rpm.
2. Prepare plasmid from overnight culture with a
Qiagen HiSpeed Plasmid Midi kit, eluting with
nuclease-free water.
3. Quantify vector by visual comparison to a High DNA
Mass Ladder run on a 1% agarose gel stained with
0.5 mg ml1 ethidium bromide. If vector is too dilute
(>100 mg ml1), concentrate the sample with a
speedvac.
193
4. Blunt cut 1 mg of vector with 10 units of HincII. Heat
inactivate the reaction.
5. Allow the sample to equilibrate to 37  C.
6. Add Antarctic Phosphatase reaction buffer to a final
concentration of 1  and 5 units of Antarctic
Phosphatase. Following dephosphorylation, heat
inactivate the sample.
7. Allow the sample to cool to room temperature.
8. Add 10  T4 DNA ligase Buffer containing ATP to a
final concentration of 1  and 2 units of ligase. Allow
the reaction to proceed for 2 h at room temperature
prior to heat inactivation.
9. Separate circular and linearized vector on a 1% agar-
ose gel stained with 0.5 mg ml1, running the sample
adjacent to a 1 kb Plus DNA ladder.
10. Extract the linearized band from the gel with a clean
razor blade, using care to minimize UV exposure of
the DNA.
11. Purify vector DNA from the agarose gel using a
MinElute Gel Extraction Kit, eluting with nuclease-
free water.
12. Quantify the vector DNA by visual comparison to a
High DNA Mass Ladder run on a 1% agarose gel
stained with 0.5 mg ml1.
13. Vector may be stored frozen at 80  C.
P. aeruginosa PAO1 Genomic DNA 4 kb Insert
Preparation
1. Inoculate 100 ml of LB media with P. aeruginosa PAO1
and allow to incubate overnight at 37  C, with shaking
at 225 rpm.
2. Extract genomic DNA using 500/G Genomic-tips and
Genomic DNA Buffer Set.
3. Quantify the genomic DNA by visual comparison to a
High DNA Mass Ladder run on a 1% agarose gel
stained with 0.5 mg ml1 EtBr.
4. Partially digest 5 mg of DNA with 10 units each of
blunt-cutting RsaI and HaeIII for 1 h.
5. Add 10% SDS to the reaction to a final concentration
of 1.
6. Resolve DNA fragments on a 1% low melting
point agarose gel stained with 0.5 mg ml1 EtBr,
running the sample adjacent to a 1 kb Plus DNA
ladder.
7. Excise with a clean razor blade the section of the gel
containing the 4 kb fragments of DNA, using caution to
minimize UV exposure of the DNA.
8. Recover DNA using a GELase Agarose Gel-Digesting
Preparation.
9. Quantify and verify length of the genomic DNA frag-
ments by visual comparison to a High DNA Mass
Ladder run on a 1% agarose gel stained with 0.5 mg
ml1 EtBr.
194 Techniques | Genome Sequence Databases: Genomic, Construction of Libraries
Ligation and Transformation
1. Following the directions included in the UltraClone
DNA Ligation & Transformation Kit, ligate insert
DNA to vector with a 3:1 insert to vector ratio.
2. Heat inactivate the reaction.
3. Precipitate the ligation by adding to the reaction 2 ml
yeast tRNA (1 mg ml1), 28 ml ultrapure water, and
100 ml 100% ethanol.
4. Vortex the mixture and allow it to cool at 20  C for
15 min.
5. Pellet by centrifuging for 15 min (13 000 rpm and
4  C).
6. Carefully remove the supernatant.
7. Wash the pellet with 70% ethanol.
8. Centrifuge the sample again (13 000 rpm and 4  C).
9. Remove the supernatant and allow the pellet to air
dry.
10. Resuspend the pellet in 2 ml of ultrapure water.
11. Transform the precipitated ligation reaction into
electrocompetent E. cloni cells included in the kit
following the suggestions of the supplier.
12. Allow cells to recover for an hour at 37  C with
shaking at 225 rpm.
13. Make a 1/100 dilution of the transformed cells prior
to plating.
14. Plate transformed cells and dilution onto YT agar
plates containing 100 mg ml1 carbenicillin.
15. Incubate the plates overnight at 37  C.
16. Count the number of colonies on the plates onto
which the dilution was plated. Calculate the number
of transformants based on this count.
Calculating the Number of Recombinant Clones
Needed for a 4000 bp Library
1. For a representational library (99.9% probability of con-
taining any given DNA sequence), calculate the number
of transformants needed using the Clarke–Carbon equa-
tion (see ‘Determining the number of transformants
needed for coverage of an entire genome’). The size of
the P. aeruginosa PAO1 genome is 6 264 404 bp.
lnð1 – P Þ
N¼
lnð1 – f Þ
with P ¼ 0.999 and f ¼ 4000/6 264 404, the number of
transformants needed N ¼ 10 815.
Confirmations
1. Fill ten sterile 15 ml conical tubes with 5 ml of LB
supplemented with 100 mg ml1 carbenicillin.
2. Pick ten colonies chosen at random from the plated
library transformation to inoculate the ten conical
tubes.
3. Allow the cultures to incubate overnight at 37  C and
225 rpm.
4. Prepare the vectors from these cultures using a
QIAprep Spin Miniprep Kit.
5. Vector DNA may be run on a 1% agarose gel to check
for appropriately sized insert or be sequenced with
primers designed to extend into the multiple cloning
site.
See also: Chromosome, Bacterial; DNA Restriction and
Modification; DNA Sequencing and Genomics; Plasmids,
Bacterial; Recombinant DNA, Basic Procedures
Further Reading
Adkins S and Burmeister M (1996) Visualization of DNA in agarose gels
as migrating colored bands: Applications for preparative gels and
educational demonstrations. Analytical Biochemistry 240(1): 17–23.
Choi KH, Kumar A, and Schweizer HP (2006) A 10-min method for
preparation of highly electrocompetent Pseudomonas aeruginosa
cells: Application for DNA fragment transfer between chromosomes
and plasmid transformation. Journal of Microbiological Methods
64(3): 391–397.
Clewell DB (1972) Nature of Col E 1 plasmid replication in Escherichia
coli in the presence of the chloramphenicol. Journal of Bacteriology
110(2): 667–676.
del Solar G, Giraldo R, Ruiz-Echevarrı́a MJ, Espinosa M, and Dı́az-
Orejas R (1998) Replication and control of circular bacterial plasmids.
Microbiology and Molecular Biology Reviews 62(2): 434–464.
Godiska R, Patterson M, Schoenfeld T, and Mead DA (2005) Beyond
pUC: Vectors for cloning unstable DNA. In: Kieleczawa J (ed.) DNA
Sequencing: Optimizing the Process and Analysis, pp. 55–75.
Sudbury, MA: Jones and Bartlett.
Grundemann D and Schomig E (1996) Protection of DNA during
preparative agarose gel electrophoresis against damage induced by
ultraviolet light. Biotechniques 21(5): 898–903.
Kues U and Stahl U (1989) Replication of plasmids in Gram-negative
bacteria. Microbiology Reviews 53(4): 491–516.
Lander ES and Waterman MS (1988) Genomic mapping by fingerprinting
random clones: A mathematical analysis. Genomics 2(3): 231–239.
Lynch MD and Gill RT (2006) Broad host range vectors for stable
genomic library construction. Biotechnology and Bioengineering
94(1): 151–158.
McClelland M (1981) The effect of sequence specific DNA methylation
on restriction endonuclease cleavage. Nucleic Acids Research
9(22): 5859–5866.
Mead DA, Patterson MK, Schoenfeld T, et al. (2002) High stability
vectors for cloning unstable DNA. Genome Sequencing and Analysis
Conference XIV. Boston, MA.
Meyer R, Figurski D, and Helinski DR (1975) Molecular vehicle properties
of the broad host range plasmid RK2. Science 190(4220): 1226–1228.
Pieterse B, Quirijns EJ, Schuren FH, and van der Werf MJ (2005)
Mathematical design of prokaryotic clone-based microarrays. BMC
Bioinformatics 6: 238.
Rand KN (1996) Crystal violet can be used to visualize DNA bands
during gel electrophoresis and to improve cloning efficiency. Elsevier
Trends Journals Technical Tips OnlineT40022.
Ravin NV and Ravin VK (1999) Use of a linear multicopy vector based on
the mini-replicon of temperate coliphage N15 for cloning DNA with
abnormal secondary structures. Nucleic Acids Research 27(17): 13.
Rodriguez RL and Denhardt DT (1988) Vectors: A survey of molecular
cloning vectors and their uses. In: Rodriguez RL and Denhardt DT
(eds.) Biotechnology. Stoneham, MA: Butterworth Publishers.
Rosche WA, Trinh TQ, and Sinden RR (1995) Differential DNA
secondary structure-mediated deletion mutation in the leading and
lagging strands. Journal of Bacteriology 177(15): 4385–4391.
 Techniques | Genome Sequence Databases: Genomic, Construction of Libraries 195

Sambrook J, Fritsch EF, and Maniatis T (1989) Molecular Cloning: A Relevant Websites
Laboratory Manual. 2nd edn. Cold Spring Harbor: Cold Spring
Harbor Laboratory Press. http://www.lucigen.com – Lucigen Corporation
Trinh TQ and Sinden RR (1991) Preferential DNA secondary structure
http://www.qiagen.com – Qiagen
mutagenesis in the lagging strand of replication in E. coli. Nature
352(6335): 544–547.
Zhu H and Dean RA (1999) A novel method for increasing the
transformation efficiency of Escherichia coli-application for bacterial
artificial chromosome library construction. Nucleic Acids Research
27(3): 910–911.