ASTM E1285-89(2000)

Standard Guide for Identification of Bacteriophage Lambda ([lambda]) or Its DNA

SCOPE
1.1 This guide covers the procedures for identifying bacteriophage lambda used in biotechnology.
1.2 There are hundreds of lambda variants that can be used for biotechnology. These lambda variants are derived from wild type lambda and differ in genome size and genotype.
1.3 If the bacteriophage lambda is to be used to construct a recombinant molecule, then the same criteria as prescribed in Section 5 should be used to characterize the newly made DNA.

General Information

• Status: Historical
• Publication Date: 09-May-2001
• ICS: 07.100.10 - Medical microbiology
• Technical Committee: E48 - Bioenergy and Industrial Chemicals from Biomass

Relations

• Replaces: ASTM E1285-89(1994) - Standard Guide for Identification of Bacteriophage Lambda ([lambda]) or Its DNA
• Effective Date: 10-May-2001
• Replaced By: ASTM E1285-01 - Standard Guide for Identification of Bacteriophage Lambda (λ) or Its DNA
• Effective Date: 10-May-2001
• Referred By: ASTM E2363-23 - Standard Terminology Relating to Manufacturing of Pharmaceutical and Biopharmaceutical Products in the Pharmaceutical and Biopharmaceutical Industry
• Effective Date: 10-May-2001

Standards Content (Sample)

NOTICE: This standard has either been superseded and replaced by a new version or discontinued.
Contact ASTM International (www.astm.org) for the latest information.
Designation: E 1285 – 89 (Reapproved 2000)
Standard Guide for
Identification of Bacteriophage Lambda (l) or Its DNA
This standard is issued under the fixed designation E 1285; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (e) indicates an editorial change since the last revision or reapproval.
INTRODUCTION
This guide is intended to determine the identification of bacteriophage lambda or its DNA. The
objective is to describe laboratory characterization procedures that are sufficient to verify that a
biological preparation believed to contain lambda or lambda DNA for use in any step of a
biotechnology process actually does contain this bacteriophage or its DNA.
This guide assumes a basic knowledge of virology and molecular biology.
1. Scope non-contractile tail about 150 nm long, ending in a single tail
fiber.
1.1 This guide covers the procedures for identifying bacte-
3.2 The genome of lambda consists of a single molecule of
riophage lambda used in biotechnology.
linear double-stranded DNA with a length of about 49 kilobase
1.2 There are hundreds of lambda variants that can be used
pairs for wild type lambda. The ends of the genome are
for biotechnology. These lambda variants are derived from
cohesive; DNA molecule is terminated by single-stranded
wild type lambda and differ in genome size and genotype.
regions of complementary base sequence allowing circulariza-
1.3 If the bacteriophage lambda is to be used to construct a
tion of a molecule. The sequence of the entire phage genome
recombinant molecule, then the same criteria as prescribed in
has been determined.
Section 5 should be used to characterize the newly made DNA.
3.3 The naturally preferred host is Escherichia coli K12.
2. Terminology
The wild type phage makes turbid plaques. Many variants,
however, have mutations in the cI gene encoding repressor.
2.1 Definitions:
These variants produce clear plaques.
2.1.1 bacteriophage—a virus that infects bacteria.
3.4 Bacteriophage lambda are used primarily as DNA vec-
2.1.2 induction—the relief of repression of transcription of
tors for cloning DNA fragments. These vectors have been
lysogenic phage genes encoding the functions for lytic growth,
engineered to accept easily the foreign DNA. The DNA
so that the phage will grow lytically.
sequences of many vectors have been altered from the wild
2.1.3 lysogen—a bacterial strain that has a phage stably
type, that is, whole (nonessential) regions have been deleted.
maintained. In the case of lambda, the phage is integrated into
Wild type lambda DNA, when cut with restriction enzymes, is
the host genome. The integrated phage is called a prophage.
used also as molecular weight markers in polyacrylamide or
2.1.4 multiplicity of infection—the ratio of infecting phage
agarose gel electrophoresis.
to host bacteria.
2.1.5 temperate bacteriophage—a bacteriophage that can
4. Bacteriophage Growth and Purification
grow lytically, killing the host, or can exist stably in the host.
4.1 Phage can be grown by any one of a number of
2.1.6 vector—a fragment of DNA usually containing an
published protocols, as follows:
origin of replication that is engineered to accept a foreign piece
4.1.1 Phage can be grown lytically by infecting a host at a
of DNA.
multiplicity of infection of usually less than one. Infection
2.1.7 wild type—the naturally occurring, original isolate.
++
requires magnesium (Mg ). The culture is grown until lysis is
3. General Information
evident (cell debris will be seen in the culture), usually several
hours. Chloroform is added to kill remaining unlysed cells and
3.1 Bacteriophage lambda is a temperate bacteriophage with
the bacterial debris is centrifuged out. The phage remains in the
an icosahedral head about 50 nm in diameter. There is a single,
supernatant fraction.
4.1.2 Phage can be grown by inducing a phage lysogen. The
more widely used lambda clo
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