1、Designation:E1285 06Standard Guide forIdentification of Bacteriophage Lambda(l)or Its DNA1This standard is issued under the fixed designation E1285;the number immediately following the designation indicates the year oforiginal adoption or,in the case of revision,the year of last revision.A number in
2、 parentheses indicates the year of last reapproval.Asuperscript epsilon()indicates an editorial change since the last revision or reapproval.INTRODUCTIONThis guide is intended to determine the identification of bacteriophage lambda or its DNA.Theobjective is to describe laboratory characterization p
3、rocedures that are sufficient to verify that abiological preparation believed to contain lambda or lambda DNA for use in any step of abiotechnology process actually does contain this bacteriophage or its DNA.This guide assumes a basic knowledge of virology and molecular biology.1.Scope1.1 This guide
4、 covers the procedures for identifying bacte-riophage lambda used in biotechnology.1.2 There are hundreds of lambda variants that can be usedfor biotechnology.These lambda variants are derived fromwild type lambda and differ in genome size and genotype.1.3 If the bacteriophage lambda is to be used t
5、o construct arecombinant molecule,then the same criteria as prescribed inSection 5 should be used to characterize the newly made DNA.2.Referenced Documents2.1 ASTM Standards:2E1873 Guide for Detection of Nucleic Acid Sequences bythe Polymerase Chain Reaction Technique3.Terminology3.1 Definitions:3.1
6、.1 bacteriophagea virus that infects bacteria.3.1.2 inductionthe relief of repression of transcription oflysogenic phage genes encoding the functions for lytic growth,so that the phage will grow lytically.3.1.3 lysogena bacterial strain that has a phage stablymaintained.In the case of lambda,the pha
7、ge is integrated intothe host genome.The integrated phage is called a prophage.3.1.4 multiplicity of infectionthe ratio of infecting phageto host bacteria.3.1.5 temperate bacteriophagea bacteriophage that cangrow lytically,killing the host,or can exist stably in the host.3.1.6 vectora fragment of DN
8、A usually containing anorigin of replication that is engineered to accept a foreign pieceof DNA.3.1.7 wild typethe naturally occurring,original isolate.4.General Information4.1 Bacteriophage lambda is a temperate bacteriophage withan icosahedral head about 50 nm in diameter.There is a single,non-con
9、tractile tail about 150 nm long,ending in a single tailfiber.34.2 The genome of lambda consists of a single molecule oflinear double-stranded DNAwith a length of about 49 kilobasepairs for wild type lambda.The ends of the genome arecohesive;DNA molecule is terminated by single-strandedregions of com
10、plementary base sequence allowing circulariza-tion of a molecule.The sequence of the entire phage genomehas been determined.34.3 The naturally preferred host is Escherichia coli K12.The wild type phage makes turbid plaques.Many variants,however,have mutations in the cI gene encoding repressor.These
11、variants produce clear plaques.34.4 Bacteriophage lambda are used primarily as DNA vec-tors for cloning DNA fragments.These vectors have beenengineered to accept easily the foreign DNA.The DNAsequences of many vectors have been altered from the wildtype,that is,whole(nonessential)regions have been d
12、eleted.Wild type lambda DNA,when cut with restriction enzymes,is1This guide is under the jurisdiction of ASTM Committee E55 on Manufactureof Pharmaceutical Products and is the direct responsibility of Subcommittee E55.04on General Biopharmaceutical Standards.Current edition approved Nov.1,2006.Publi
13、shed November 2006.Originally in1989.Last previous edition approved in 2001 as E1285 01.DOI:10.1520/E1285-06.2For referenced ASTM standards,visit the ASTM website,www.astm.org,orcontact ASTM Customer Service at serviceastm.org.For Annual Book of ASTMStandards volume information,refer to the standard
14、s Document Summary page onthe ASTM website.3Hendrix,R.,Roberts,J.,Stahl,F.,and Weisberg,R.,Lambda II,Cold SpringHarbor Laboratory,Cold Spring Harbor,NY,1983.1Copyright ASTM International,100 Barr Harbor Drive,PO Box C700,West Conshohocken,PA 19428-2959,United States.Copyright ASTM International Prov
15、ided by IHS under license with ASTM Licensee=Ohio State University/5967164005,User=ahmadi,rozitaNot for Resale,03/26/2012 04:35:27 MDTNo reproduction or networking permitted without license from IHS-,-,-used also as molecular weight markers in polyacrylamide oragarose gel electrophoresis.3Mice trans
16、genic for bacterio-phage lambda have been constructed to enable mutationdetection in the mouse genome.4,55.Bacteriophage Growth and Purification5.1 Phage can be grown by any one of a number ofpublished protocols,3as follows:5.1.1 Phage can be grown lytically by infecting a host at amultiplicity of infection of usually less than one.Infectionrequires magnesium(Mg+).The culture is grown until lysis isevident(cell debris will be seen in the culture),usually severalhours.Chloroform is added to kill
