Help: Cloning a 60KB fragment - (Nov/01/2010 )

Hi guys,

I need clone a 60KB fragment (a gene island) from a bacteria strain and put it into E. Coli. Just wondering if there is any good cloning protocol/kit available specially for it? I found online an "expand long template pcr system" from Roche that provides a kit for 5-20 kb fragment, which is not long enough apparently. Is there any kit that could cover 60kb in a single cloning step. ...

In addition, if I want to put the fragment into E.Coli, it seems that Bacterial artificial chromosome is a commonly used vector. Could you suggest any BAC vector that I can get/buy online?

Thank you very much.
Mobo

You should be able to clone into one of the BAC vectors. Commonly available ones include pFOS1 and pBelobac11. Probably the most straightforward way of doing this is randomly cloning large fragments of the genome into BACs and testing for the presence of your desired insert with pcr screening. Multiplexing many samples will reduce the number of pcr reactions necessary to identify the desired clones. You'll have to screen both ends of your insert separately.

Check out the Copycontrol fosmids and the pIndigoblue pre-digested plasmids from Epicentre at http://epibio.com

Thanks you SO MUCH, Phage434! The vectors you suggested and the website link are extremely helpful!

I'm a kind of newbe to BAC cloning and just wondering if you could elaborate a bit about "randomly cloning large fragments of the genome into BACs" and "testing for the presence of your desired insert with pcr screening" as well as "Multiplexing" ? Are there are references/online materials about that? Another thing is that, how difficult it is to do this cloning?

Thanks a lot.

Best,
Mobo

phage434 on Mon Nov 1 19:38:58 2010 said:

So, you'd basically be producing a BAC library of DNA fragments of your bacterial genome. Keywords to look for in protocols are library construction. Usually, this is done by preparing high molecular weight genomic DNA (phenol chloroform extraction is a good way). The DNA is then fractionated, with either rare cutting enzymes, partial digestion, or mechanical techniques such as needle shearing or nebulation. The ends are repaired (see Epicenter and NEB for end repair enzymes) and then cloned into a BAC vector. You want to try to make > 60 Kb fragments, which is somewhat of a challenge, at several levels. This is not a good first project.

You can screen BACs by pcr of short fragments at each end of the region you are cloning (assuming you have sequence sufficient for primers).

Multiplexing is simply the idea that instead of screening each individually, you can screen groups of say 8 at a time by mixing plasmid DNA from 8 preps and doing pcr on the mixture.

How big is your genome? Do you really need a single clone containing all of it?