Overnight cultures (5 ml) are split into two 500 µl fractions for storage at −70☌ and a 4-ml aliquot for plasmid DNA purification. XL1-Blue is far superior for Qiagen preps, for instance, than DH5 or JM109 strains. The host strain chosen depends in part on the plasmid preparation method used. An aliquot of the packaged pBlue script is mixed with host cells (generally XL1–Blue) and plated on LB plates supplemented with 50 µg ml −1 ampicillin. The supernatant from the in vivo excision is stable for at least a year at 4☌ without loss of titer.įresh colonies are plated weekly for preparation of plasmid DNA templates for sequencing. Colonies obtained from plating of the rescued library are analysed for insert size and compared to insert sizes from phage plaques obtained by polymerase chain reaction (PCR) to be certain that small clones were not preferentially amplified in the rescue. A successful in vivo excision generally gives a high titer of packaged pBlue script and only a minimal increase in the percentage of blue colonies compared with blue plaques from the amplified phage library. While the standard protocols from Stratagene are used, great care is given to determining the optimum ratio of host cells, helper phage and cDNA-containing lambda phage. The amplified library is rescued en masse by in vivo excision into pBlue script packaged as single-stranded circles in M13 phage heads. The packaged library is amplified in Escherichia coli SURE cells. Other protocols are presented in chapters in this book. cDNA is ligated and packaged essentially according to the manufacturer's (Stratagene) instructions in the Uni-ZAP and Gigapack gold kits.
Computional analysis may also take many forms based around sequence similarity searching and compositional characteristics.ĬDNA libraries are often made in λ-ZAP, using the directional cloning system with EcoRI/XhoI arms. Many variations on this theme are possible, depending upon the specific application, such as subtraction or screening of the library to bias sequencing toward particular groups of clones.
At each step, relevant lab techniques including mRNA purification, cDNA library construction, clone selection, and automated sequencing are shown. The building blocks of an EST project are listed with key decision points.