Genomic Cloning Technical Manual

Genomic Cloning Technical Manual

An optimal strategy for genomic cloning should meet three requirements: 1) a maximum number of recombinants should be obtained with a minimal background of nonrecombinants, 2) the genomic library constructed should be representative of genomic DNA sequences, and 3) the strategy should facilitate restriction mapping of the cloned DNA. Promega's preparations of LambdaGEM ^® -11 and EMBL Vector Arms are optimized for the highest recombinant efficiencies and lowest nonrecombinant backgrounds possible. When using Promega's Bam H I Lambda Arms, nonproductive ligations of genomic DNA with the central stuffer fragment are extremely rare (<100pfu/µg arms, or one per 10 ⁵ recombinants). Thus, more recombinant DNA is cloned and larger libraries are generated, while fewer filters need to be processed per screening experiment. Because of these low backgrounds, the need for Spi selection agains
点击浏览该文件 t parental phage in recombination-proficient ( rec +) hosts is eliminated.

An ultra-low background cloning strategy can be used with Promega's genomic cloning vectors. Genomic DNA partially digested with Mbo I or Sau 3A I can be cloned into dephosphorylated Bam H I arms (EMBL3 or LambdaGEM ^® -11 Vectors). These vectors accept DNA fragments ranging in size from 9-23kb. However, for library constructions using these Bam H I sites, the genomic DNA must be size fractionated following the partial digestion step to avoid any possibility of cloning two or more genomic fragments into the same site. For this purpose, only fragments greater than 14kb should be selected for ligation with the vector. Background production of parental phage is low because religation of these arms with the central stuffer fragment has been eliminated by secondary digestion with Eco R I and removal of the small Bam H I- Eco R I fragments.