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Gigapack III Packaging Extracts - Details & Specifications

  • Gold: Recombinant lambda phage with the highest packaging efficiency available, to achieve maximal primary library size. Absence of restriction activity prevents the degradation of methylated DNA for a more representational library
  • Plus: Recombinant lambda phage when the highest packaging efficiency is not required
  • XL: Recombinant lambda phage with selectivity for phage containing large inserts

 

Packaging extracts are used to package recombinant lambda phage with high efficiency. The single-tube format of Gigapack III packaging extract simplifies the packaging procedure and increases the efficiency and representation of libraries constructed from highly methylated DNA. Each packaging extract is restriction minus (HsdR McrA McrBC McrF Mrr) to optimize packaging efficiency and library representation. When used in conjunction with restriction-deficient plating cultures, Gigapack III packaging extract improves the quality of DNA libraries constructed from methylated DNA.

Because choice of an in vitro packaging extract designed to achieve specific experimental goals is important, we offers three different packaging extracts: Gigapack III Gold packaging extract, Gigapack III Plus packaging extract, and Gigapack III XL packaging extract. Gigapack III Gold packaging extract produces the highest packaging efficiency commercially available (2 × 109 pfu/µg) and is designed for use in cDNA and genomic library construction. Gigapack III Plus packaging extract is an economical alternative to Gigapack III Gold packaging extract when the highest packaging efficiency is not required. Gigapack III XL packaging extract preferentially packages large inserts (i.e., 47–51-kb recombinants), which eliminates the need for time-consuming size fractionation steps and the loss associated with sizing columns or sucrose gradients. This packaging extract is designed for use in genomic lambda and cosmid library construction.

Optimal packaging efficiencies are obtained with lambda DNAs that are concatemeric. Ligations should be carried out at DNA concentrations of 0.2 µg/µL or greater, which favors concatemers and not circular DNA molecules that only contain one cos site. DNA to be packaged should be relatively free from contaminants. Polyethylene glycol (PEG), which is contained in some ligase buffers, can inhibit packaging. The volume of DNA added to each extract should be between 1 and 4 µL. To obtain the highest packaging efficiency [i.e., the number of plaque-forming units per microgram (pfu/µg) of DNA], package 1 µl of the ligation reaction and never more than 4 µL. Increased volume (i.e., >4 µL) yields more plaque-forming units per packaging reaction, but fewer plaque-forming units per microgram of DNA.

DNA that is digested with restriction enzymes and re-ligated packages less efficiently (by a factor of 10–100) than uncut lambda DNA. For example, uncut wild-type lambda DNA packages with efficiencies exceeding 1 × 109 pfu/µg of vector when using a Gigapack III packaging extract. However, predigested arms, when ligated to a test insert, yield ~5 x 106 – 1 x 107 recombinant plaques/µg of vector.

For Research Use Only. Not for use in diagnostic procedures