For generating genomic and cDNA libraries, bacteriophage lambda has been successfully used as a host vector for quite some time. Efficient libraries have been generated primarily because of the high efficiency with which the phage particles infect the bacteria in vitro
. Different packaging systems, which can be used to package recombinant lambda phage with high efficiency, are now widely available. This high infection efficiency of lambda has also led to the construction of different variants of the original lambda vector, such as cosmids and fosmids.
Stratagene’s Gigapack® III XL Packaging Extract has been designed to produce highly efficient packaging (2x109 pfu/µg) and can be used for both cDNA and genomic library construction. I have used this system often for constructing highly size selective genomic libraries containing large inserts in my cosmid vector. The single tube format of the Gigapack® packaging extract simplifies the packaging procedure and increases the efficiency and representation of libraries constructed from highly methylated DNA. The extract is easy to use as it contains all the components and buffers needed to perform the packaging reaction in a single tube. One just has to add the ligation reaction in it. Each packaging extract is restriction minus (HsdR-McrA-McrBC-McrF-Mrr-) to optimize packaging efficiency and library representation. The constructed genomic libraries from methylated DNA have improved quality when used in conjunction with restriction-deficient host strains.
The Gigapack® III XL packaging extracts preferentially package large inserts (i.e. 47-51 kb recombinants), which eliminates the need for time-consuming size fractionation steps and the subsequent loss associated with sizing columns or sucrose gradients. Other variants of this packaging extract are also available which can give higher packaging efficiencies but are less selective of insert size.
Stratagene has designed the extracts for construction of both cosmid and phage libraries with optimal packaging efficiencies obtained after concatamer formation. A cosmid or a lambda vector has typical cos sites in the vector. During ligation reaction, long concatamers are formed which are recognized by the packaging proteins during packaging, and a certain size length of DNA gets packaged into the phage head. The size selection is a big help because it reduces the total number of required clones in a given library. In the case of cosmids, after packaging and transfection, the cosmid along with the insert goes into the bacterium and gets circularized in the form of a plasmid which is very easy to manipulate in downstream processes. So ligation should be carried out at DNA concentrations of 0.2 µg/µl or greater, which favors concatamers and not circular DNA molecules that only contain one cos site.
The steps in the given protocol are very easy to follow. I do not recommend any modifications when using a cosmid vector along with Stratagene’s Gigapack® III XL Packaging Extract. For preparation of bacterial cultures for infection, I recommend to strictly follow the procedure stated in the protocol provided. For transfection, do not use overnight cultures because they often reduce the titers after infection because dead cells may be there which may bind to recombinant phages and decrease the successful infection rate. There is no sample ligation reaction protocol within the manual so one has to be really careful in selecting the amount of DNA to be ligated. I recommend a 4-6 times higher insert to vector ratio in a ligation reaction while taking care that the total amount does not increase to more than 0.2-0.3 µg/µl DNA concentration. Use of PEG normally added in the quick ligation kits, is not recommended as it can promote blunt-ended fragments by macromolecular crowding. In addition, PEG is inhibitory to the packaging reactions. One more thing to consider is the volume of the ligation reaction. For using Stratagene’s Gigapack® III XL Packaging Extracts, one should be aware that only 1-4 µl of the ligation reaction can be used for packaging. So it is imperative to use concentrated DNA solutions for ligation in small volumes. This has two benefits: more concatamer formation and fewer circular DNA products, and more recombinants.
For titration of phage and cosmid libraries, the protocol is excellent and can also be used as a help with other packaging systems that lack a good protocol. I have used this extract many times and have gotten good results. With optimized ligation conditions, which one would have to evaluate for oneself, this system is quite successful in producing recombinant phages, whether to maintain in phage libraries or as cosmid clones. Stratagene provides E.coli VCS257 as the host strain, a derivative of DP50 supF and should be used for plating the packaged test DNA. The supF mutation in the bacterial host strain helps to increase the plating efficiency and stability of the recombinants. It does not allow color selection of recombinants or IPTG induction of expression. And if color selection is required then I recommend E.coli HB101 as the host strain. A lambda strain, λc1857, is also provided as a positive control, so one can check the actual packaging efficiency of the extracts.
In conclusion, Stratagene’s Gigapack® III XL Packaging Extract is very useful and convenient for constructing cosmid or phage libraries. There are other variants of the system available as Gigapack® III Gold Packaging Extract and Gigapack® III Plus Packaging Extract.