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Breaking the 109 Efficiency Barrier One challenge when searching for novel genes in a cDNA library is that rare transcripts occur infrequently, making it extremely important to construct the most representative cDNA libraries possible. Gigapack® packaging extracts were the first lambda extracts to break the 1 x 109 pfu/ug packaging efficiency barrier. This extremely high efficiency allows you to more easily build representative libraries and find your rare transcripts (Figure 1).
A key factor in increasing efficiency is eliminating restriction enzyme systems from the extract. Early lambda packaging extracts often carried restriction enzyme systems that were capable of cleaving foreign DNA and had a significantly negative effect on the efficiency of DNA cloning. We eliminated the most problematic systems when developing the Gigapack family of extracts, which helped achieve high-packaging efficiency. Over the years, we have continued to improve the Gigapack extract family, removing further restriction enzyme systems and developing Gigapack extracts that are optimized for specific applications, such as packaging large recombinants (47 to 51 kb).
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The In Vivo Excision Revolution Not only did we break the efficiency barrier, but we also revolutionized lambda’s ability to be used in downstream applications. Even though lambda has an extremely high packaging efficiency, lambda DNA is difficult to use in sequencing, site-directed mutagenesis, and other post-cloning manipulations. The cloned cDNA is best suited in a plasmid vector when performing these types of applications. Researchers were forced to either subclone their insert into a plasmid or forego the efficiency lambda offered. The Lambda ZAP® vector system eliminated this subcloning step, allowing easy excision of the pBluescript® phagemid. It is easy to perform sequencing, blue/white color selection, and protein synthesis in E. coli with pBluescript. The excision is not only easy, but can be done on a mass scale, enabling you to go from a lambda library to a plasmid library in one step (Figure 2).
Lambda Today cDNA cloning is an extremely important method for isolating clones and discovering new genes. Because sequencing projects for organisms such as humans and mice have been completed, many genes have been discovered. However, for researchers discovering unique genes from other organisms, lambda libraries are still extremely valuable. Gigapack packaging extracts and Lambda ZAP vectors remain the highest quality, most innovative products available for cloning into lambda libraries.
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