Phage Lamda Manipulation Protocols

Alkaline Phosphatase Treatment of Bacteriophage lambda Vector DNA Protocol Removal of the 5'-phosphate groups from the internal termini of bacteriophage {lambda} arms is used to prevent self-ligation and suppress the background of nonrecombinant bacteriophages during cloning.

Assaying the DNA Content of Bacteriophage lambda Stocks and Lysates by Gel Electrophoresis Protocol Protocol describes the DNA content of bacteriophage lambda stocks can be easily and rapidly estimated.

Expression of Cloned Genes in E. coli Using the Bacteriophage lambda pL Promoter Protocol Protocol describes how (1) to clone cloned sequences encoding open reading frames in plasmids carrying bacteriophage {lambda} pL promoters, (2) to optimize expression of target proteins in transformants carrying these recombinants, and (3) to scale-up production of foreign proteins.

Extraction of Bacteriophage lambda DNA from Large-scale Cultures Using Formamide Protocol Formamide can be used instead of proteinase K to dissociate bacteriophage {lambda} coat proteins from the viral {lambda}. The procedure is rapid and works best with large-scale preparations of bacteriophage {lambda}.

Extraction of Bacteriophage lambda DNA from Large-scale Cultures Using Proteinase K and SDS Protocol DNA is best isolated from bacteriophage lambda particles by digesting the viral coat proteins with a powerful protease such as proteinase K, followed by extraction with phenol:chloroform. This procedure is easily adapted for use with smaller-scale preparations of bacteriophage lambda.

Identifying DNA-binding Proteins in Bacteriophage ambda Expression Libraries Protocol Protocol describes how to identify cloned cDNAs encoding proteins that bind to specific DNA sequences. The methods used are very similar to those used for immunological screening of expression libraries except that the nitrocellulose filters carrying immobilized proteins are screened with 32P-labeled double-stranded DNA rather than with antibodies.

Lambda DNA Preparation Protocol Protocol describes a plate method that gives very good yield for cloning. Includes: T-TYN Media + Mg+2; T-TYN Plates; T-TYN Top Agarose.

Lambda gt22a Screening Protocol Protocol for lambda gt22a screening. Includes: T-TYN Media + Mg+2; T-TYN Plates; T-TYN Top Agarose; Phage Dilution Buffer (PDB); 10X Kinase Buffer (-DTT); Calf Thymus DNA.

Ligation of Bacteriophage lambda Arms to Fragments of Foreign Genomic DNA Protocol Pilot ligations and packaging reactions are used to establish the amounts of fragmented genomic DNA and bacteriophage {lambda} arms that yield the maximum number of recombinants. Additional ligation and packaging reactions may then be set up to yield a comprehensive library of genomic DNA.

Preparation of Bacteriophage lambda DNA Cleaved with a Single Restriction Enzyme Protocol Bacteriophage {lambda} vectors that allow genetic selection of recombinant bacteriophages (e.g., the EMBL series, {lambda}2001, {lambda}DASH, {lambda}ZAP, and {lambda}gt10) can be prepared for cloning by simple digestion by one or more restriction enzymes.

Preparation of Bacteriophage lambda DNA Cleaved with Two Restriction Enzymes Protocol Many replacement vectors (e.g., the EMBL series, {lambda}2001, and {lambda}DASH) contain a series of restriction sites, arranged in opposite orientations, at each end of the central stuffer fragment. Digestion of these vectors with two different restriction enzymes yields left and right arms, a stuffer fragment, and short segments of the polycloning sites. These can easily be removed from the arms by differential precipitation with isopropanol or spun-column chromatography.

Rapid Analysis of Bacteriophage lambda Isolates: Purification of lambda DNA from Plate Lysates Protocol is used to purify small amounts of bacteriophage {lambda} DNA that are suitable for use as substrates for restriction enzymes and templates for DNA and RNA polymerases.