BAC DNA extraction for PacBio

[juckdnarocks]

Hi,

I want to extract DNA from BAC for PacBio RS sequencing, is there any protocols or kits for extraction ?

Any suggestions are welcome.

[nextomics]

I have the same task, do you finished your project?

[krobison]

I don't know about BACs & PacBio specifically, but for cosmids we find that Plasmid Safe is very useful to add upstream of Illumina sequencing -- it removes the linear genomic fragments. With PB in particular, you really don't want to waste sequence on them.

This item may be of interest to you

http://www.broadinstitute.org/software/allpaths-lg/blog/?p=641

[Olaf Blue]

Plasmid Safe has been available since 1993 and has been used in all sorts of plasmid preps for years. For BAC preps, there are a number of products available for BAC purification from E. coli and all use a modified Maniatis-based alkaline lysis method. The key is to be super-gentle after addition of the "Solution 2" or alkali/SDS buffer so that the genomic and BAC shearing are kept to a minimum. The main concern is with yields as BACs are "mandatory" single copy plasmids (unless having been made in Epicentre's CopyControl system) and thus larger liquid cultures may be needed (>50 ml).

Cheers, Olaf