Phage isolation
and purification

Clone library
construction

Sequencing

Assembly

Post-assembly
analyses

There are multiple steps where precious CsCl purified phage DNA will be needed during the process from phage particle to phage genome. First, if phage DNA is not limiting it would be worth using ~10 ng to size the DNA using PFGE (Steward et al. 2000, Wommack et al. 1999). Second, phage DNA will be required for the construction of clone libraries -- conventional methods require >1000 ng DNA while novel advances in clone library construction is guaranteed to provide >100,000 random clones from <1-10 ng DNA. Finally, significant amounts of DNA (~1,000 ng) might be required for closing the genome.

Because of the difficulties in getting significant DNA from phage that are difficult to culture and the potential for modified bases and/or toxic genes that inhibit conventional cloning techniques, the construction of RASLs or LASLs might be required. RASL clone library construction is described in Rohwer et al (2001) below. More information about custom cloning to make LASL clone libraries can be found at http://www.lucigen.com/custom/index.html and http://www.sci.sdsu.edu/PHAGE/LASL/index.htm.

Rohwer F, Seguritan V, Choi DH, Segall AM, Azam F. 2001. Production of shotgun libraries using random amplification.
Biotechniques. Jul;31(1):108-12, 114-6, 118. -- PDF

Steward, G., J. Montiel, and F. Azam. 2000. Genome size distributions indicate variability and similarities among marine viral assemblages from diverse environments. Limnol Oceanogr 45:1697-1706.

Wommack, K. E., J. Ravel, R. T. Hill, J. S. Chun, and R. R. Colwell. 1999. Population dynamics of Chesapeake Bay virioplankton: total community analysis by pulsed-field gel electrophoresis. Appl Environ Microbiol 65:231-240.