dna sequencing protocols

CHAIVER 1
DNA Sequencing
Hugh G. Grifin and Annette M. Grifin
1. Introduction
Methods to determine the sequence of DNA were developed in the
late 1970s (1,2) and have revolutionized the science of molecular
genetics. The DNA sequences of many different genes from diverse
sources have been determined, and the information is stored in interna-
tional databanks such as EMBL, GenBank, and DDBJ. Many scientists
now accept that sequence analysis will provide an increasingly use-
ful approach to the characterization of biological systems. Projects are
already underway to map and sequence the entire genome of organisms
such as
Escherichia coli, Saccharomyces cerevisiae, Caenorhabditis
elegans,
and
Homo sapiens.
In the recent past, large-scale sequenc-
ing projects such as these were often dismissed as prohibitively expen-
sive and of little short-term benefit, while DNA sequencing itself was
seen as a repetitive and unintellectual pursuit. However, this view is
now changing and most scientists recognize the importance of DNA
sequence data and perceive DNA sequencing as a valuable and often
indispensable aspect of their work. Recent technological advances,
especially in the area of automated sequencing, have removed much
of the drudgery that used to be associated with the technique, and
modern innovative computer software has greatly simplified the analy-
sis and manipulation of sequence data. Large-scale sequencing
From Methods m Molecular Biology, Vol. 23. DNA Sequencmg Protocols
Edited by. H. and A. Gnffm Copyright Q1993 Humana Press Inc., Totowa, NJ
1

2 Griffin and Griffin
projects, such as the Human Genome Project, produce the DNA sequen-
ces of many unknown genes. Such data provide an impetus for molec-
ular biologists to apply the techniques of reverse genetics to produce
probes and antibodies that can be used to identify the gene product, its
cellular location, and its time of appearance in the developing cell (3).
A function can be assigned by mutant analysis or by comparison of
the deduced amino acid sequence with proteins of known function.
Therefore, DNA sequencing can act as a catalyst to stimulate future
research into many diverse areas of science.
The two original methods of DNA sequencing described in 1977 (1,2)
differ considerably in principle. The enzymatic (or dideoxy chain termina-
tion) method of Sanger (I) involves the synthesis of a DNA strand from
a single-stranded template by a DNA polymerase. The Maxam and
Gilbert (or chemical degradation) method (2) involves chemical degra-
dation of the original DNA. Both methods produce populations of radioac-
tively labeled polynucleotides that begin from a fixed point and terminate
at points dependent on the location of a particular base in the original
DNA strand. The polynucleotides are separated by polyacrylamide
gel electrophoresis, and the order of nucleotides in the original DNA
can be read directly from an autoradiograph of the gel (4).
Although both techniques are still used today, there have been many
changes and improvements to the original methods. While the chemi-
cal degradation method is still in use, the enzymatic chain termina-
tion method is by far the most popular and widely used technique for
sequence determination. This process has been automated by utilizing
fluorescent labeling instead of radioactive labeling (Chapters 33-37),
and the concepts of polymerase chain reaction (PCR) technology have
been harnessed to enable the sequencing reaction to be “cycled” (Chap-
ters 21, 26, and 34). Other recent innovations include multiplexing

(Chapter 28), sequencing by chemiluminescence rather than radioac-
tivity (Chapter 29), solid phase sequencing (Chapter 25), and the use of
robotic work stations to automate sample preparation and sequenc-
ing reactions (Chapter 38).
2. Maxam and Gilbert Method
In the original Maxam and Gilbert method (2) a fragment of
DNA
is radiolabeled at one end and then partially cleaved in four different
chemical reactions, each of which is specific for a
particular base or
DNA Sequencing
3
type of base. This results in four populations of labeled polynucleo-
tides. Each radiolabeled molecule extends from a fixed point (the
radiolabeled end) to the site of chemical cleavage, which is determined
by the DNA sequence of the original fragment. Since the cleavage is
only partial, each population consists of a mixture of molecules, the
lengths of which are determined by the base composition of the origi-
nal DNA fragment. The four reactions are electrophoresed in adjacent
lanes through a polyacrylamide gel. The DNA sequence can then be
determined directly from an autoradiograph of the gel. The original
method has been improved over the years (5). Additional chemical
cleavage reactions have been devised (6), new end-labeling techniques
developed (7,8), and shorter, simplified protocols have been produced
(Chapter 32). The main advantage of chemical degradation sequenc-
ing is that sequence is obtained from the original DNA molecule and
not from an enzymatic copy. It is therefore possible to analyze DNA
modifications such as methylation, and to study protein/DNA interac-
tions. Chemical sequencing also enables the determination of the DNA
sequence of synthetic oligonucleotides. However, the Sanger method

is both quicker and easier to perform and must remain the method of
choice for most sequencing applications.
3, Sanger Method
The Sanger (or chain termination) method (I) involves the synthe-
sis of a DNA strand from a single-stranded template by a DNA poly-
merase. The method depends on the fact that dideoxynucleotides
(ddNTPs) are incorporated into the growing strand in the same way
as the conventional deoxynucleotides (dNTPs). However, ddNTPs
differ from dNTPs because they lack the 3’-OH group necessary for chain
elongation. When a ddNTP is incorporated into the new strand, the
absence of the hydroxyl group prevents formation of a phosphodiester
bond with the succeeding dNTP and chain elongation terminates at
that position. By using the correct ratio of the four conventional dNTPs
and one of the four ddNTPs in a reaction with DNA polymerase, a
population of polynucleotide chains of varying lengths is produced.
Synthesis is initiated at the position where an oligonucleotide primer
anneals to the template, and each chain is terminated at a specific
base (either A, C, G, or T depending on which ddNTP was used). By
using the four different ddNTPs in four separate reactions the com-
4
Griffin and Griffin
plete sequence information can be obtained. One of the dNTPs is
usually radioactively labeled so that the information gained by elec-
trophoresing the four reactions in adjacent tracks of a polyacryla-
mide gel can be visualized on an autoradiograph.
The original method used the Klenow fragment of DNA polymerase
I to synthesize the new strands in the sequencing reactions, and this
enzyme is still used today (Chapter 12). Other enzymes such as
Sequenase (Chapter 14), T7 polymerase (Chapter 13), and
Taq

poly-
merase (Chapter 15) are also widely used. Each enzyme has its own
particular properties and qualities, and the choice of polymerase will
depend on the type of template and the sequencing strategy employed.
4. Templates for DNA Sequencing
The polymerase reaction requires single-stranded template. This is
usually achieved by utilizing Ml3 phage that can produce large
amounts of just one strand of DNA as part of its normal replicative
cycle. Double stranded (replicative form) Ml3 can also be isolated, and
this is used to clone the DNA fragment to be sequenced. The qualrty
of DNA sequence data achieved using Ml3 template is extremely good
and many researchers prefer to subclone to Ml3 prior to sequencing.
Sequencing reactions can be performed directly on plasmid DNA,
the double stranded molecule being denatured prior to sequencing.
Recent innovations in DNA purification techniques and the availability
of improved polymerases have greatly enhanced the quality of data
produced by plasmid sequencing methods (Chapters 14, 18, and 19).
Sequence determination can also be performed directly on cosmid
clones (Chapter 21), lambda clones (Chapter 20), and on PCR prod-
ucts (Chapters 23-25). In particular, the advent of cycle sequencing (Chap-
ter 26) has vastly increased the range of templates that can be used.
5. Sequencing Strategies
A lot of sequencing performed is confirmatory sequencing to check
the orientation or the structure of newly constructed plasmids, or to
determine the sequence of mutants. This type of sequencing can be
easily achieved by subcloning a restriction fragment into Ml3 and
sequencing using the universal primer. Alternatively, a custom-designed
oligonucleotide primer can be synthesized and sequencing performed
without the need for any subcloning.
DNA Sequencing

5
The determination of long tracts of unknown sequence, however,
requires careful planning and the utilization of one of a variety of strate-
gies including: the shotgun approach, directed sequencing strategies,
and the gene walking technique. A random, or shotgun, approach
involves subcloning random fragments of the target DNA to an appro-
priate vector such as Ml3 (Chapter 7). Sequences from these recombi-
nants are determined at random until the individual readings can be
assembled into a contiguous sequence. This is achieved using a
sequence assembly computer program (9,lO). The disadvantage of
this method is the redundancy in the sequence data obtained, each
section of DNA being sequenced several times over. However, the
strategy benefits from making no prior assumptions about the DNA
to be sequenced, such as base composition or the presence of certain
restriction sites.
Directed strategies usually involve the construction of a nested set
of deletions of the fragment to be sequenced. Progressive deletions
of the fragment are generated with a nuclease, each deletion being approx-
imately 200-300 bp. Following deletion the fragments are recloned
into Ml3 or a plasmid vector adjacent to the universal primer site.
The subclones are then sequenced in order of size, with the sequence
of each clone overlapping slightly with the one before. In this way, a
large tract of contiguous sequence is determined on one gel. The disad-
vantage is the labor and time involved in constructing the deletions.
Several methods are available for deletion construction including the
use of exonuclease III (Chapter 8), T4 DNA polymerase (Chapter 9),
and DNase I (Chapter 10). It is essential to sequence both strands of
the DNA and this usually entails generating two sets of deletions.
Perhaps the simplest method of sequencing is the gene walking
technique (Chapter 11). This involves the initial sequencing of approx-

imately 200-400 bp of the end of a cloned fragment using the univer-
sal primer (the sequence of the other end can be achieved with the
reverse primer). This sequence information is then used to design a
new oligonucleotide primer, which will provide the sequence of the
next 200-400 bp, and so on across the entire length of the insert. This
method is the least labor intensive because no deletion construction
or generation of random clones is necessary, and template DNA can
be made in the one batch since the template is the same for all sequenc-
ing reactions. However, the delay involved in synthesizing a new oli-
Griffin and Griffin
gonucleotide primer before the next reaction can be performed may
considerably prolong the time taken to sequence a long tract of DNA.
The cost of oligonucleotide synthesis may also be prohibitive.
6. Automation in DNA Sequencing
One of the major advances in sequencing technology in recent years
is the development of automated DNA sequencers. These are based
on the chain termination method and utilize fluorescent rather than
radioactive labels. The fluorescent dyes can be attached to the sequenc-
ing primer, to the dNTPs, or to the terminators, and are incorporated
into the DNA chain during the strand synthesis reaction mediated by
a DNA polymerase (e.g., Klenow fragment of DNA polymerase I,
Sequenase, or
Taq
DNA polymerase). During the electrophoresis of
the newly generated DNA fragments on a polyacrylamide gel a laser
beam excites the fluorescent dyes. The emitted fluorescence is collec-
ted by detectors and the information analyzed by computer. The data
are automatically converted to nucleotide sequence. Several such
instruments are now commercially available and are becoming increas-
ingly popular (11; Chapters 33-37).

Other aspects of the sequencing procedure that are being automated
include template preparation and purification, and the sequencing
reactions themselves. Robotic workstations are currently being devel-
oped to perform these tasks (Chapter 38).
7. Cycle Sequencing
Cycle sequencing is a new and innovative approach to dideoxy
sequencing. Its advantages over conventional sequencing techniques
are that the reactions are simpler to set up, less template is required,
the quality and purity of template are not as critical, and virtually any
single- or double-stranded DNA can be sequenced (including lambda,
cosmid, plasmid, phagemid, M13, and PCR product). In this method,
a single primer is used to linearly amplify a region of template DNA
using
Taq
polymerase in the presence of a mixture of dNTPs and a
ddNTP. Either radioactive or fluorescent labels can be used, making
cycle sequencing technology as relevant to automated processes as it
is to manual methods (Chapters 26, 34-36).
As in conventional dideoxy sequencing methods, cycle sequenc-
ing involves the generation of a new DNA strand from a single-stranded
DNA Sequencing
template, synthesis commencing at the site of an annealed primer,
and terminating on the incorporation of a ddNTP. The difference is
that the reaction occurs not just once but 20-30 times under the control
of a thermal cycler (or PCR machine). This results in more and better
sequence data from less template. The process of denaturing a double-
stranded molecule is eliminated, with denaturation occurring auto-
matically in the thermal cycler. The development of cycle sequencing
techniques has made a major contribution to DNA sequencing meth-
odology, improving the reliability and efficiency of DNA sequence

determination and eliminating time-consuming steps.
8. Aim of This Book
The purpose of this book is to provide detailed practical proce-
dures for a number of DNA sequencing techniques. Although proto-
cols for DNA sequencing methods are available elsewhere, there was
a need for a book that comprehensively covered the vanguard tech-
niques now being applied in this rapidly evolving field. Each contri-
bution is written so that a competent scientist who is unfamiliar with
the method can carry out the technique successfully at the first attempt
by simply following the detailed practical procedures that have been
described by each author.
Even the simplest techniques occasionally go wrong, and for this rea-
son a “Notes” section has been included in most chapters. These notes
will indicate any major problems or faults that can occur, their sources,
and how they can be identified and overcome. Since the purpose of this
book is to describe practical procedures and not to go into great depth
regarding theory, a comprehensive reference section is included in
most chapters, enabling the reader to refer to other publications for
more detailed theoretical discussions on the various techniques.
References
1. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) DNA sequencing wrth chain-
termmator inhibitors. Proc. Natl. Acad. Sci USA 74, 5463-5467.
2. Maxam, A. M and Gilbert, W (1977) A new method for sequencing DNA
Proc. Natl. Acad. Scl USA 74,560-564.
3. Barrell, B. (1991) DNA sequencmg: present limitations and prospects for the
future. FASEB J 5,40-45
4. Sambrook, J., Frrtsch, E F., and Maniatrs, T. (1989) Molecular Cloning. ,4
Laboratory Manual 2d ed , Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, NY.
Griffin and Griffin

5 Maxam, A. M. and Gilbert, W. (1980) Sequencing end-labeled DNA with base-
specific chemical cleavages. Meth. Enzym. 65,499-560.
6. Ambrose, B J. B. and Pless, R. C. (1987) DNA sequencing. Chemical meth-
ods. Meth. Enzymol. 152,522-538.
7. Volckaert, G. (1987) A systematic approach to chemical DNA sequencing by
subcloning in pGV451 and derrved vectors. Meth Enzym. 155,23 l-250
8 Eckert, R. L. (1987) New vectors for rapid sequencing of DNA fragments by
chemical degradation. Gene 51,247-254.
9. Dolz, R. (1993) Fragment assembly programs, in DNA sequencmg: Computer
Analysis ofSequence Data, (Griffin, A. M. and Grrffm, H. G., eds.), Humana
Press, Totowa, NJ. (Ch. 2).
10 Staden, R. (1992) Managing sequencing projects, m DNA sequencing. Com-
puter Analysis of Sequence Data, (Griffin, A. M. and Griffin, H. G., eds.),
Humana Press, Totowa, NJ. (Ch 17).
11 Hunkapiller, T , Karser, R J., Kopp, B F , and Hood, L. (1991) Large-scale
and automated DNA sequence determination. Science 254,59-67
&IAE’TER
2
Ml3 Cloning Khicles
Their Contribution to DNA Sequencing
Joachim Messing
1. Introduction
For studies in molecular biology, DNA purification has been essen-
tial, in particular for DNA sequencing, probing, and mutagenesis.
The amplification of DNA in E. coli by cloning vehicles derived from
M13mp or pUC made expensive physical separation techniques like
ultracentrifugation unnecessary. Although today the polymerase chain
reaction is a valuable alternative for the amplification of small DNA
pieces (I), it cannot substitute for the construction of libraries of DNA
fragments. Therefore, E. coli has served not only as a vehicle to

amplify DNA, but also to separate many DNA molecules of similar
length and the two DNA strands simultaneously. For this purpose, a
bacteriophage like M 13 can be used. The various viral
cis-
and
truns-
acting functions are critical not only for strand separation, but also to
separate the single-stranded DNA from the
E. coli
cell by an active
transport mechanism through the intact cell wall.
Although it may have been somewhat surprising to some how many
changes in its DNA sequence the phage tolerated, manipulations of
this amplification and transport system have been extended today even
to the viral coat proteins for the production of epitope libraries (2).
Much of the work is now more than a decade old, but experience has
confirmed the usefulness of some simple biological paradigms. Tech-
From Methods m Molecular Biology, Vol 23’ DNA Sequencmg Protocols
Edlted by. H and A Gnffm Copyright 01993 Humana Press Inc , Totowa, NJ
9
10 Messing
niques that were new and limiting fifteen years ago included auto-
mated oligonucleotide synthesis and the use of thermostable enzymes,
which add a critical dimension to molecular biology today. Neither
necessarily replaces the previous techniques, but they create greater
flexibility, enormously accelerate scientific investigations, and even
make certain analyses possible for the first time.
However, DNA sequencing of larger contigs (several overlapping
sequences that can be linked) have benefited from economizing on
the synthesis of new oligonucleotides (3,4). Even in the absence of

automated oligonucleotide synthesis in the earlier years, the concept
of a universal primer could be developed by alternative techniques.
2. Development of DNA Sequencing Techniques:
A Discussion
In 1974, at one of the first meetings on the use of restriction endo-
nucleases in molecular biology some of these ideas became clear,
Work on the chemical synthesis of a tRNA gene was presented, and
the initial work on sequencing phage $X174 using restriction frag-
ments as primers for the plus-minus method was discussed. At that
time, oligonucieotide synthesis required a major effort and could not
easily be generally applied. Restriction fragments offered an alterna-
tive. They could be used as primers for DNA synthesis in vitro and
for marker-rescue experiments to link genetic and physical maps of
viruses like SV40, both forerunners for DNA sequencing and site-
directed mutagenesis.
There were several reasons to use @X 174 as the first model in devel-
oping DNA-sequencing techniques and determining the sequence of
an entire autonomous genome. First, it was one of the smallest DNA
viruses; it is even smaller than M13. Second, the mature virus con-
sists of single-stranded DNA, eliminating the need to separate the
two strands of DNA for template preparation; this is even more criti-
cal if one wishes to use double-stranded restriction fragments as prim-
ers. Third, a restriction map was superimposed on the genetic map by
marker-rescue experiments (5). The latter feature still serves today as
a precondition for other genomes. Restriction sites were critical as
signposts along the thousands of nucleotides and provided the means
to dissect the double-stranded replicative form or RF of @X174 in
small, but ordered pieces that permitted the DNA sequencing effort
Ml3 Cloning Vehicles
11

to proceed in a walking manner along the genome. Today the restric-
tion map can be replaced by any DNA sequence (e.g., STS), since the
synthesis of oligonucleotides is so rapid that researchers can use the
DNA sequence that was just read from a sequencing gel to design
and produce an oligonucleotide to extend the sequencing gel further
in the 5’ direction. Therefore, the use of oligonucleotides instead of
restriction fragments in such a primer walking method would have
enormously accelerated the @X174 project.
At the Cleveland Conference on Macromolecules in 198 1, after a
talk that I had given, the replacement of shotgun sequencing by such
a method was suggested by a colleague, who, as a pioneer in DNA syn-
thesis and its automation, saw a perfect match of this emerging technol-
ogy with DNA sequencing. Another expert in the chemical synthesis
of DNA, Michael Smith, had more interest in the applications than
improving the method, and had switched from the diester method to the
phosphoramidite method (6), as well as conceiving the idea of using
oligonucleotides in marker rescue (7). These innovative researchers
pioneered marker rescue and established the physical and genetic map
of $X174 (5). It is clear that today’s protein engineering had its roots
right there with the right people at the right time, because they also
recognized that oligonucleotides could eliminate the need for strand
separation for DNA sequencing (8), and developed this method until
it reached greater maturity (4,9). Even double-stranded DNA sequenc-
ing with universal primers became easier with the development of
the pUC plasmids (IO).
Despite all the advantages of choosing @X174 as a model system,
Sanger’s group nearly picked a different single-stranded DNA phage,
fd. In principle, E. coli has two different types of single-stranded DNA
phage, represented by $X174 and fd. The first is packaged into an
icosahedral head; it kills and lyses the host cell, but does not require

F pili, which are receptor sites on the surface of the cell wall encoded
by F factors. Its host range is restricted to E. coli C. Phage like fd can
only infect male-specific E. cd, producing pili at their surface that
are packaged in a filamentous coat and discharged from the cell with-
out lysis; infected cells can continue to divide. These differences are
critical, but there was another reason for choosing fd originally, The
major coat protein encoded by gene VIII of the phage, a very small
but very abundant protein, had been sequenced by protein sequen-
12 Messing
cing methods. Therefore it seemed obvious, particularly to those who
had pioneered protein sequencing, to use protein sequence to check the
DNA sequence. The protein sequence allowed the design and synthe-
sis of an oligonucleotide that would prime in vitro DNA synthesis
within the coat protein gene. Furthermore, the derived DNA sequence
had to match the protein sequence. Of course, the codon redundancy
of many amino acids made it difficult to design a unique primer, and
it might have been not too surprising that the approach did not lead to
the correct DNA sequence (11). It turned out later that this was not
caused by the choice of codons, but to a mistake in the protein sequence
instead, Still, the paradigm of reverse genetics again has its roots
right there.
3. Replication Systems and Ml3
In 1974, a research group at the Max Planck Institute of Biochem-
istry in Munich became interested in viruses, initially in
E.
coli more
than in mammalian cells. The “Abteilung” was organized in sub-
groups, and one subgroup was interested in developing in vitro reph-
cation systems using both bacteriophage and small plasmids. This
had a strong biochemical emphasis, and the researchers rapidly began

to learn about single-stranded DNA replication. Eleven years earlier,
Hofschneider had isolated a similar filamentous phage from the
Munich sewers that he named after a series of phage with the initial
M (12). Number 13 was the one that was studied most. Looking for a
different research topic than plasmids or DNA replication, the possi-
bility of combining Ml3 phage production with the in vitro DNA
synthesis-based method of DNA sequencing was seen.
Although this might have been obvious to those familiar with phage
replication, innovative methods were needed for adaptation to DNA
cloning techniques. The walking method for sequencing $X174 was
the strategy used at that time, and some thought that it would be dif-
ficult to clone large fragments into Ml3 (although the author’s record
was around 40 kb) and that a walking method might therefore have a
limited use. Logically, the only alternative to the walking method
was the use of shotgun clomng and a universal primer. The replica-
tive form of Ml3 could be used to clone DNA fragments of a size
slightly larger than necessary for single sequencing reactions, and a
universal sequence near the cloning site would be used as a primer.
Ml3 Cloning Vehicles
This would shift the work from preparing primers to preparing tem-
plates, which still remains more economical (13). If they were numer-
ous, cloning was much faster than any biochemical technique, and
with these thoughts in mind, a plan took shape to construct in vitro
recombinants of phage Ml3 without using existing methods. One
might recall that in vitro recombinants were usually based on drug-
resistance markers. This led to the development of plasmid vectors
with unique cloning sites that were scattered ail over the plasmid
genome (14).
4. Transposons Mutagenesis
Both Zinder’s and Schaller’s laboratory had in mind, and actually

later used, transposons to develop f 1 and fd transducing phage (15,16).
However, one could predict that such a course of experiments,
although useful for plasmid cloning vehicles, would be less useful
for M13. It seemed plausible, and such an experiment could demon-
strate that, in contrast to $X174, filamentous phage can accommo-
date additional DNA by extending the filamentous coat; infected cells
can be treated like plasmid-containing cells. To some degree this had
already been proved since one group had already described mutants
of more than unit length (17). Another advantage of transposon
mutagenesis was that insertion mutants would be naturally selected.
This was one of the biggest obstacles from the beginning. Although
plasmids and bacteriophage h were natural transducing elements, fila-
mentous phage had never been shown to have this property, and it
was not obvious whether insertion mutants would be viable. This was
difficult because it was already known that amber mutants of most
viral genes not only cause abortive infection, but also lead to killing
of the host, something that does not happen when these mutations are
suppressed. Therefore, insertion mutants that can be used as plas-
mids would kill the cell.
It was thus predictable that insertion mutants had to be restricted
to noncoding regions. Therefore, a decision was made to use a restric-
tion enzyme that recognized at least two different sites in the intergenic
region of the RF. Rather than asking whether the intergenic region
contains a target site for transposons, the restriction map showed that
it was possible to get at least two different insertion mutants within
the intergenic region. The only difficulty in such an experiment was
14 Messing
to find conditions where the restriction enzyme would cut RF only
once at any of the possible target sites, so that a population of unit
length RF could be ligated to the appropriate marker DNA fragment.

However, there was another reason not to use drug-resistance mark-
ers. Infected cells still divide, but very slowly. Therefore, selection
takes much longer than with plasmids, but it makes it very easy to
distinguish infected from noninfected cells on a bacterial lawn. A
single infection grown on a bacterial lawn forms a turbid plaque. If
bacterial cells are then transfected by the calcium chloride technique
of Mandel and Higa (18), a transformed cell can be recognized as a
plaque. Hence, no selection technique is necessary, but the ability to
distinguish between wild-type M 13 and M 13 insertion mutants would
remain a problem. Although it was quite plausible to think of the
histochemical screen used for bacteriophage hplac by Malamy et al.
(19), the
la&
gene would have been a large insertion. However, it
turned out that, rather than using entire genes as markers, one could
clone only the portion encoding the amino-terminal and the repress-
ible control region, and provide the rest in
tram
by the host of the
phage. This became clear when Landy et al. (20) wrote on the purifi-
cation of an 800-bp Hind11 fragment from
hplac
capable of a-comple-
mentation in a cell-free transcription-translation system.
An informal sequence of this fragment showed that it was 789-bp
long and included the first 146 codons of the
ZacZ
gene, but it was
still necessary to assemble many components and purify several
restriction endonucleases. Work began after some strains and puri-

fied Zac repressor were traded; this allowed the purification of the
789-bp Hind11 fragment out of about fifty other restriction fragments
by simply filtering the DNA/lac repressor complex through a nitro-
cellulose filter. After adding IPTG, it was possible to recover the DNA
in solution. Using DNA-binding proteins for purifying and cloning
promoter regions is now a well established technique, but ligating
restriction fragments via blunt ends was not established at the time
when this experiment was ready. As described elsewhere (21), only
two transformants were obtained-one of them was saved and named
M 13mp 1. Electron microscopy proved that added DNA was packaged
as a filamentous phage and produced as single-stranded DNA (22,23).
Now the path took a more formal shape. Not only would the histo-
chemical screen work by detecting a blue among colorless plaques, but
Ml3 Cloning Vehicles 15
1 2 3 4 5 6 7 8 9 10 WlJmpl
ATG ACC ATG ATT AC- TC A CTG GCC GTC (+ or Viral strand)
GGmAT TC
(+ or viral strand)
CT TA AG (- or complementary strand)
12 3 4 5 6 7 8 9 10 M13mp2
ATG ACC ATG ATT ACE AAT TC
A CTG GCC GTC (+ or Viral strand)
EcoRI
Fig. 1. Creation of an EcoRI sue by chemical mutagenesis. By screening the
nucleotides of the first ten codons of the 1ucZ gene, we found that the sequence
GGATTC could erther be converted into an EcoRI site GAATTC or a BamHI sue
GGATCC by a single base change. Since there already was a BumHI site in gene
III, but no EcoRI site m M13mp1, and I had an ample supply of EcoRI enzyme
purified myself, we decided to select the GAATTC site that also changed codon
GAT for asparttc acid to AAT for asparagine (24).

it could be reversed. One uncertainty was how to introduce new restric-
tion sites
in the right
region,
Such a site had to be unique for M 13mp 1
and positioned not somewhere in the viral genome, but in the
ZacZ
region, so that insertion mutants would not give rise to a-comple-
mentation. Inspection of the sequence showed that there were not
many sequences in the amino-terminal region that could be converted
in a single step into a unique restriction site. Attempts to use EcoRI
linkers that became available at the time to “marker rescue” them
did not succeed. Without somebody to synthesize oligonucleotides
that were more homologous to the
Zac
region, it was hopeless.
As an
alternative, a chemical mutagen was tried. It was known that methy-
lated G could mispair with uracil or thymine, therefore, by methylat-
ing the single-stranded Ml3 DNA with nitrosomethylurea, a mutation
could be introduced into the minus strand and the subsequent RF
molecules (Fig. 1).
Unfortunately, there was no good genetic selection for this proce-
dure. It would require brute-force methods of enriching EcoRI-sensi-
tive RF from a transformed phage library by gel electrophoresis of
linear versus circular molecules. Still, it was hard to believe when
this author isolated RF that was not only sensitive to EcoRI, but had
exactly the predicted base change in codon
5
of the

1acZ
gene (24).
16 Messing
The same mutagenesis led to two more EcoRI mutants and a mutant
RF that was resistant to BumHI, a site within gene III. At that time,
there was still much concern that many mutations might not be toler-
ated because of changes in the protein sequence or the secondary
structure of RNA. Therefore, changes in the lac DNA should prob-
ably occur at a higher frequency than in the viral DNA. Furthermore,
the aminoterminus of the
1acZ
gene appeared to be more flexible since
it was demonstrated that fusion proteins retained P-galactosidase func-
tion. On the other hand, this author did not recognize the tremendous
selection power for suppressor mutations. In other words, any muta-
tion that was introduced could potentially be compensated for by
another mutation somewhere else. The primary mutant might give a
low titre, but because of the growth advantage a suppressor mutant
would rapidly take over. An example of such a case is M 13mp 1, Later,
Dotto and Zinder (25) showed that insertion mutants at the mpl Hue111
site gave a low titre phenotype: Since M13mpl gave a normal titer,
they searched for a suppressor mutation. Codon 40 in gene II of
M 13mp 1 was indeed changed.
5. The Need for a Universal Primer
Researchers continue to use chemical mutagens to get rid of restric-
tion sites. The reason for this becomes clear when they return to the
use of Ml 3 in DNA sequencing. The EcoRI site in M13mp2 allowed
cloning by screening for colorless plaques. Now one could readily
purify these plaques and prepare a template for sequencing the inserted
DNA. Still, as with the $X174 project, a restriction fragment from

the adjacent Zuc DNA needed to be purified as a primer, and such a
fragment was subcloned into a plasmid for convenience (26). Such a
primer fragment still needed to be denatured since it was double-
stranded, and had to be cut off after the sequencing reaction to pro-
duce a shift of the 3’ end in the sequencing gel.
Although such a protocol could still be improved on, a more seri-
ous obstacle arose suddenly from the concern over the biological con-
tainment of Ml 3 recombinant DNA. The NIH Recombinant Advisory
Committee (RAC) thought that the conjugation proficient E. coli host
strains could lead to the spread of Ml3 infection and pose a risk in
using Ml3 as a cloning vehicle. On the other hand, using one of the
truD
or
tru1
mutants reduces conjugation by a factor of 106, but they
Ml3 Cloning Vehicles 17
still were infectious to M13. Since this F factor carrying the traD muta-
tion was wild-type lac, a histochemical screen with the mp vectors
would not be possible, and one would have to return to drug-resistant-
type M 13 vectors. The scientific reasoning of RAC is hard to under-
stand. First, nobody argued against Agrobacterium tumefaciens as a
plant transformation vector, although it was conjugation proficient
and could easily spread in the environment. Second, F pili were never
made under stress or anaerobic conditions, something that was already
known as “phenocopies.” Conjugation in the human gut was in any
case nearly zero. Third, Ml3 infection per se reduces conjugation by
a factor of 106. It was hard to argue with so many well known scien-
tists at that time, so a new series of E. coli strains (JM series) all carrying
the Ml5 deletion on the F’ traD36 episome was constructed. Since it
was a concern of NIH, it was necessary to summarize the status of all

the strains for potential users in the NIH Bulletin (27).
Although DNA sequencing of eukaryotic origin by Ml3 cloning
was now possible, preparation of the primer from the plasmid was
still cumbersome. It was clear that an oligonucleotide to replace the
restriction fragment as a universal primer was needed. Inquiries
regarding the synthesis of a universal primer by a commercial sup-
plier revealed that the cost of such an attempt made it out of the ques-
tion A further attempt and timely support produced success, not only
with a universal primer but also with another application of oligo-
nucleotides (3).
In 1978 this author made another interesting observation, namely
that in-frame insertions of linkers in the EcoRI site could still give a
positive color reaction, One of these isolates, called M13mp5, could
be used to clone both EcoRI and Hind111 fragments at the same site
and with the same primer for sequencing (27). The utility of creating
cloning sites on top of each other was based on the universal primer
concept, but in turn caused the development of multiple cloning sites
(MCS) or polylinkers that are now found in all cloning vehicles and
provide many additional uses (Fig. 2). Therefore, work began on the
synthesis of an oligonucleotide that could be inserted into the EcoRI
site and generate restriction sites recognized by six basepair cutters
like BamHI, AccI,
SmaI,
or Hind11 useful for cloning either blunt-
ended fragments or fragments with sticky ends produced by four base
cutters like
Sau3A, TaqI,
and
HpaII (Fig. 3) (consistent with a DNA
18 Messing

A
Kanr
ECORI-BamHI-SalI-PstI-SalI-BamHI-EcoRI
AccI
AccI
HincII
HincII
B
EcoRI-SmaI-BamHI-SalI-PstI-Hind111
Imp8 1
XmaI AccI
HincII
HindIII-PstI-SalI-BamHI-SmaI-EcoRI (mpg 1
AccI XmaI
HincII
5 I
>3 ’ ~xxxxxxxxxxxxxxxxxxx~
GAATTCCCGGG
<
exoII1 GCCAAGCTT
CTTAAGGGCC- < exoVII > ACGEGGTTCGAA
BarnHI PstI
Fig. 2. Symmetric (a) and asymmetric (b) polylinkers. Two other “tricks” were
used m the construction of polylmkers. One type of polylmker was symmetrical
where all sites except the central one occurred twice. By cloning a drug-resistance
marker into the central site, the polylinker could be used in a linker scanning method
of coding regions (10) and (34). The other type of polylinker was a pair, where two
vectors contain an array of sites only once, but each of them m the opposite onen-
tation Cloned DNA no longer could be cloned out with a single enzyme as in the
first type, but DNA could be cloned by using two different sites at the same time.

This had the advantage that the orientation of a cloned fragment could be deter-
mined. By using a vector pair, both orientations can be obtained with the same pair
of restriction cuts and therefore each strand of a restriction fragment could become
the viral strand of Ml3 and available as a template for sequencing Furthermore,
by using two restriction enzymes that produce 3’ and 5’ overhangs, one can either
use It for cloning oligonucleotlde hbrarles or to generate umdlrectional deletions
with exonuclease III
shotgun sequencing approach). This also required a renewed chemi-
cal mutagenesis to eliminate the AccI and the HincII sites naturally
occurring in M13. All single mutations were combined by marker
rescue to give rise to M13mp7 (3). This was just the system, but did
shotgun sequencing succeed?
Ml3 Cloning Vehicles 19
Unique vector sequence
G'GATC C
C CTAG'G
GT'CG AC
CA GC'TG
GTC'GAC
CAG'CTG
HincII
Compatible target sites
N'GATC N
N CTAG'N
MYbOT
NT’CG AN
NA GC'TN
NC'CG GN
NG GC'CN
NNN'NNN

NNN'NNN
Restrlction enzymes like
AALL, HaeIII, etc.;
Ba.UlorExoIIIIV;
sheared and repaired DNA
Fig. 3. Sticky and blunt-end cloning of small fragments mto unique cloning
sites. By destgning a unique sequence for the M13mp vectors that were recogmzed
by restriction enzymes that could cut a hexanucleotide sequence in various ways
by etther producing sticky ends of 4 or 2 bases, or blunt ends, the variety of DNA
fragments that could be cloned next to the umversal primers were endless Note
that the sequence GTCGAC was recognized by SufI, AccI, and H&II, each pro-
ducing different ends. In our sequencing project wtth cauliflower mosatc virus we
generated small DNA fragments for shotgun DNA sequencing by cleaving CAMV
with EcoRI*, Mb&, HpaII, TagI, HincII, HueIII, and A/u1 (28) Later we used
DNaseI (35), sonication (30), and a combination of exonuclease III and VII to
generate blunt ends (36)
Initially, lack of funds and proper laboratory facilities made a dem-
onstration impossible. Financial support finally arrived, however, and
a dedicated research team began producing the sequence of cauli-
flower mosaic virus on the side (8031 bp). This was accomplished in a
record time of three months, and the results were finally published a
year later (28).
20 Messing
6. Conclusion
During the same time, several researchers reported that they could
not finish their work on sequencing the mitochondrial DNA if they
could not use a host for Ml3 approved by the NIH guidelines. This
author gave them not only the JM strains, but also the newly devel-
oped M13mp7 for blunt-end cloning to facilitate their work. Although
this author’s work was not published, having been rejected by PNAS

as trivial at the time, in the end, I recognized that there was no sense
in competing but that the scientific community should be used as a
laboratory at large, and this indeed became reality (29).
Becoming overwhelmed by requests for strains and protocols, the
newly developing reagent companies were turned to for help. Their
educational and service role helped immensely to disseminate the
knowledge needed to train students and investigators in academia
and industry in M 13 cloning, sequencing, and site-directed mutagen-
esis (30). Along the way, the first Apple-based software on shotgun
sequencing was developed (31,32), in addition to a textbook for an
undergraduate course (33). A good overview of M13mp, pUC vec-
tors, and helper phage has also appeared (21). As with all methods of
wider scope, modifications and refinements have been produced in
many laboratories as many chapters in this book show. However, some
principles have not changed: whenever there is a way to use a cell or
parts of one as a machine, scientists seem to get ahead faster and
cheaper, and that is what biotechnology is all about.
Acknowledgment
Most of this author’s work on Ml3 was supported by the Deutsche
Forschungs Gemeinschaft, grant no. 5901-9-0386, and the Depart-
ments of Agriculture and Energy, grant no. AC0243 lER10901.
References
1. Saiki, R. K., Gelfand, D. H., Stoffel, S , Scharf, S. J., Higuchi, R., Horn, G. T.,
Mullis, K. B., and Ehrlich, H A. (1988) Primer-directed enzymatic amplifica-
tion of DNA with a thermostable DNA polymerase. Science 239,487- 491
2. Scott, J. and Smith, G. (1990) Searchmg for peptIde ligands with an epitope
library. Science 249,386 -390.
3. Messing, J., Crea, R., and Seeburg, P. H. (1981) A system for shotgun DNA
sequencing. Nucl. Acids Res. 9, 309-321.
4. Norrander, J., Kempe, T., and Messing, J (1983) Improved Ml3 vectors using

oligonucleotide-directed mutagenesis.
Gene
26, 101-106.
Ml3 Cloning Vehicles 21
5. Edgell, M. H., Hutchison, C. A., III, and Sclair, M. (1972) Specific endonuclease
R fragments of bacteriophage $X174 deoxyribonucleic acid. J. Viral. 9,574-582
6 Beaucage, S. L. and Caruthers, M H. (1981) Deoxynucleoside phosphorami-
dites-a new class of key Intermediates for deoxypolynucleotide synthesis.
Tetrahedron Lett. 22, 1859-l 862.
7. Hutchison, C. A., III, Phtlhps, S., Edgell, M. H., Gillam, S., Jahnke, P., and
Smith, M (1978) Mutagenesis at a specific position in a DNA sequence. J.
Biol. Chem. 253,655 l-6560.
8. Smith, M , Leung, D W , Gillam, S., Astell, C. R , Montgomery, D. L., and
Hall, B D (1979) Sequence of the gene for iso-1-cytochrome C in Saccharo-
myces cerevisiae. Cell 16, 753-761.
9 Zoller, M and Smith, M. (1982) Oligonucleotide-directed mutagenesis using
M13-derived vectors. an efficient and general procedure for the production of
point mutations in any fragment of DNA. Nucl. Acids Res. 10,6487-6500
IO. Vieira, J. and Messing, J (1982) The pUC plasmtds, an M 13mp7 derived sys-
tem for msertion mutagenesis and sequencing with synthetic universal prtm-
ers Gene 19,259-268.
11 Sanger, F , Donelson, J E., Coulson, A. R., Kossel, H., and Fischer, H (1973)
Use of DNA polymerase I primed by a synthetic ohgonucleotide to determine a
nucleotide sequence in phage f 1 DNA. Proc. Nat1 Acad. Ser. USA 70,1209-l 2 13
12. Hofschneider, P H (1963) Untersuchungen uber “kleine” E coli K12
Bacteriophagen M12, M13, und M20. Z. Naturforschg. Mb, 203-205.
13. Davison, A. J. (1991) Experience in shotgun sequencing a 134 kilobase pair
DNA molecule DNA Sequence 1,389-394
14 Bolivar, F , Rodriguez, R. L., Greene, P. J., Betlach, M. V., Heynecker, H L ,
Boyer, H. W , Crosa, J. W , and Flakow, S (1977) Construction and characteriza-

tion of new cloning vehicles. II. A multipurpose cloning system. Gene 2,95-l 13
15. Herrmann, R., Neugebauer, K., Zentgraf, H., and Schaller, H. (1978) Transpo-
sition of a DNA sequence determining kanamycin resistance into the single-
stranded genome of bacteriophage fd MOE Gen. Genet. 159, 17 1.
16 Vovis, G F. and Ohsumi, M. (1978) The filamentous phages as transducing
particles, m The Single-Stranded DNA Phages (Denhardt, D. T , Dressler, D.,
and Ray, D S , eds ), Cold Spring Harbor Laboratory, Cold Spring Harbor,
NY, pp 445-448
17 Salivar, W. O., Henry, T. J., and Pratt, D. (1967) Purtfication and properties of
diploid particles of coliphage M13. Virology, 32,41-5 1
18. Mandel, M. and Higa, A (1970) Calcium-dependent bacteriophage DNA mfec-
tion. J. Mol Blol., 53, 159-162.
19 Malamy, M H., Fiandt, M , and Szybalski, W. (1972) Electron microscopy of
polar insertions in the lac operon of Escherichia co11 Mol. Gen Genet 119,
207-222
20 Landy, A , Olchowski, E , and Ross, W. (1974) Isolation of a functional lac
regulatory region. Mol Gen Gene& 133,273-28 1.
2 1. Messing, J. (1991) Clonmg m Ml 3 phage or how to use biology at its best
Gene 100,3-12
22 Messing
22 Messing, J , Gronenborn, B., Muller-Hill, B., and Hofschnetder, P H. (1977)
Frlamentous cohphage Ml3 as a cloning vehtcle insertion of a HrndII frag-
ment of the fat regulatory region m the Ml3 rephcattve form in vitro Pruc
Natl. Acad. SCL USA 74,3642-3646.
23. Messing, J. and Gronenborn, B (1978) The filamentous phage Ml3 as carrter
DNA for operon fusions in vitro, m The Single-Stranded DNA Phages
(Denhardt, D T., Dressler, D., and Ray, D. S., eds.), Cold Spring Harbor Labo-
ratory, Cold Spring Harbor, NY, pp. 449-453.
24 Gronenborn, B. and Messing, J. (1978) Methylatron of single-stranded DNA
m vttro mtroduces new restrtctrons endonuclease cleavage sites. Nature 272,

375-377
25 Dotto, G. P. and Zmder, N D (1984) Reduction of the mmimal sequence for
mmatton of DNA synthesis by qualitative and quantttatrve changes of an ml-
trator protein Nature 311,279-280
26. Heidecker, G., Messmg, J , and Gronenborn, B. (1980) A versatile prrmer for
DNA sequencing m the M13mp2 clonmg system Gene 10,69-73.
27 Messing, J (1979) A multrpurpose cloning system based on the single-stranded
DNA bacteriophage M13. Recombinant DNA Technical Bulletin, NIH Publt-
canon No. 79-99,2, No. 2,43-48
28 Gardner, R. C., Howarth, A J., Hahn, P 0, Brown-Leudi, M , Shepherd, R
J., and Messmg, J. (198 1) The complete nucleotide sequence of an mfectious
clone of cauliflower mosaic virus by M13mp7 shotgun sequencmg. Nucl Acrds
Res. 9,2871-2888
29 Holden, C (1991) Briefings Science 254,28
30. Messing, J (1983) New Ml3 vectors for clonmg. Methods Enzymol. 101,20-78
31 Larson, R. and Messing, J. (1982) Apple II software for Ml3 shotgun DNA
sequencmg Nucl Acids Res 10, 39-49
32 Larson, R. and Messing, J. (1983) Apple II computer software for DNA and
protean sequence data DNA 2,3 l-35.
33 Hackett, P. H., Fuchs, J A , and Messing, J (1984) An introduction to recom-
bmant DNA techniques Basic Experiments in Gene Manzpulution Benjamin-
Cummings, Menlo Park, CA
34 Messing, J., Vieira, J , and Gardner, R (1982) Codon insertion mutagenesis to
study functional domains of P-lactamase In vrtro mutagenesis Cold Spring
Harbor Laboratory, Cold Spring Harbor, NY, pp 52
35 Messmg, J. and Seeburg, P H (1981) A strategy for hrgh speed DNA sequen-
cing, in Developmental Biology Using Purified Genes (Brown, D. and Fox, F ,
eds ), ICN-UCLA Symposia on Molecular and Cellular Biology, vol 23 Aca-
demic, NY, pp 659-669.
36 Yanisch-Perron, C , Vieira, J , and Messing, J (1985) Improved Ml3 phage

cloning vectors and host strains, nucleottde sequences of the M 13mp and pUC
vectors Gene 33,103-l 19
hAP!l’ER
3
Cloning into Ml3
Qingzhong
Yu
1. Introduction
The bacteriophage M 13 has been developed into a cloning vector
system for obtaining single-stranded DNA template required for the
dideoxy chain termination method of sequencing DNA (1,2). Gen-
eral aspects of bacteriophage Ml3 as a cloning vector system are
reviewed in Chapter 2, and the preparation of foreign DNA fragments
for Ml3 cloning is described in Chapter 7. In this chapter the prepa-
ration of Ml3 vectors and the ligation of foreign DNA fragments
(inserts) into Ml3 vectors are described.
2.
Materials
2.1. Preparation
of
Replicative Form (RF) Ml3 DNA
1. L-Broth: Bacto-tryptone l%, bacto-yeast extract O.S%, NaCl 1%.
2. 2X YT: Bacto-tryptone 1.6%, bacto-yeast extract 1%, NaClO.5%.
3. M9 minimal medium: Na,HFQ, . 7H20
12.8 g, IU-12P04 3 g, NaC10.5 g,
NH&l 1.0 g, 20% glucose 20 mL, Hz0 to 1 L.
4. BacteriophageM13: A single blue plaque from a freshly transformed plate.
5.
E. coli
JM 103 or JM 109: A colony grown on an M9 minimal agar plate.

6. Solution 1: 50 mII4 Glucose, 25 mM Tris-HCI, pH 8.0, 10 rniI4 EDTA,
pH 8.0, autoclaved and stored at 4°C.
From. Methods m Molecular Brology, Vol 23. DNA Sequencmg Protocols
E&ted by- H. and A Gnffm Copyright 01993 Humana Press Inc., Totowa, NJ
23
24 YU
7. Solution 2: 0.2M NaOH, 1% SDS, freshly mix together equal volumes
of 0.4M NaOH and 2% SDS stocks before use.
8. Solution 3: 60 mL 5M Potassium acetate, 11.5 mL glacial acetic acid,
28.5 mL H20, stored at 4OC.
9. TE: 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0.
10. Phenol-chloroform: Mix well equal volumes of TE (pH 8.0)-saturated
phenol (nucleic actd grade) and chloroform (AR grade), stored at 4°C
m a dark glass bottle.
11.
3M
NaAc, pH 5.2, stored at 4°C.
12. Ethanol: Absolute alcohol, stored at -20°C.
13. RNase: 1 mg/mL DNase-free pancreatic RNase A.
2.2. Preparation
of
Ml3 Vectors
1. Restriction enzymes.
2. 10X RE buffers.
3. Calf intestinal alkaline phosphatase (CIP).
4. 10X CIP dephosphorylation buffer.
5. Proteinase K 10 mg/mL.
6.
10% SDS. ’
7.

0.5M
EDTA, pH 8.0.
8.
3M
NaAc, pH 7.0, stored at 4°C.
9. 10X TBE: Tns base 108 g, Boric acid 55 g, EDTA 9 2H20 9.5 g, HZ0 to 1 L.
10. 0.8% Agarose gel in 1X TBE.
2.3. Ligation
of
Inserts into Ml3 Vectors
1. T4 DNA ligase.
2. 10X Ligation buffer.
3. 10 mM ATP, stored at -20°C.
4. Ml3 vectors (prepared as described in the methods
section).
5. Inserts (having termini compatible with the vectors).
All enzymes and buffers are stored at -20°C. Other solutions can
be stored at room temperature except when indicated otherwise.
3. Methods
A number of M 13 vectors have been constructed (.2,3) and are com-
mercially available from several companies. Therefore, it would be
more convenient to purchase the Ml3 vectors than
to
prepare them
oneself. Sometimes, however, you may need a Ml3 vector with a
special cloning site to fit your cloning strategy. Thus the preparation
of Ml3 vectors is described below.
Cloning into Ml3
25
3.1. Mini Preparation

of
RF Ml3 DNA
1. Inoculate 5 mL of L-Broth in a 20-mL sterile culture tube (e.g., universal)
with one bacterial colony (e.g., JM103 or JM109) from an M9 minimal
agar plate. Incubate at 37OC in an orbital shaking incubator overnight.
2. Add 50 l,tL of the bacteria culture to 2 mL of L-Broth in a 5-mL culture
tube (e.g., bijoux). Inoculate this culture with Ml3 bacteriophage by
touching a single blue plaque from a transformatton plate with a sterile
toothpick and washing its end m the culture. Incubate the infected cul-
ture at 37°C for 4-5 h m an orbital shaking incubator.
3. Transfer 1.0-l .5 mL of the culture to a microfuge tube and centrifuge at
12,OOOg for 2 min at room temperature in a microfuge. Remove supernatant
to a fresh tube, being careful not to disturb the pellet. If desired, the single
strand Ml3 (single-stranded) DNA can be prepared from the supernatant.
4. Remove any remaining supernatant by aspiration from the tube containing
the bacterial pellet. Resuspend the pellet by ptpeting it or vigorous vortex-
ing in 100 pL of solution 1 and leave at room temperature for 5-10 mm.
5. Add 200 cls, of freshly prepared solution 2. Close the tube and mix the
contents by mvertmg the tube rapidly five times. Do not vortex. Store
the tube on ice for 5 mm.
6. Add 150 pL of ice-cold solution 3. Vortex the tube gently m an inver-
ted position for 10 s. Store on ice for 5 min.
7. Centrifuge at 12,OOOg for 5 min and transfer the supernatant to a fresh tube.
8. Add an equal vol of phenol:chloroform, mix by vortexing for 20-30 s.
Spin as in step 7 and transfer the aqueous phase (top layer) to a fresh tube.
9. Add 2 vol of ethanol, mix by vortexing, and stand for 5 min at room
temperature.
10. Spin as m step 7 and remove the supernatant by gentle asptratton.
11, Wash the pellet with 1 mL of 70% ethanol. Spin for 2 mm m the same
orientation of the pellet. Remove the supernatant as m step 10. Vacuum

dry for 3-5 mm or air-dry for 10 min.
12. Dissolve the pellet m 20 pL of TE (pH 8.0) contammg RNase (20 pg/
mL) to remove RNA. Vortex briefly. The double-stranded RF Ml 3 DNA
is now ready for analysis by digestion with restriction enzymes.
3.2. Preparation
of
Ml3 Vectors
3.2.1 Digestion with a Single Restriction Enzyme (RE)
1. Digest RF Ml3 DNA with a single RE in the followmg reaction:
RF Ml3 DNA 10 $L (400 ng)
Restriction enzyme 3-5-fold excess
10X appropriate buffer 2 pL
H2O
make up to 20 pL total vol