Large DNA cloning?

[Jose E. Mejia]

In article <Pine.GSO.3.92.961008223124.10604A-100000 at chuma>, "Yi Zhang
(BIO)" <yzhang1 at chuma.cas.usf.edu> wrote:

> I am going to clone a large peice of viral DNA fragment into pPAC vector.
> This insert is 150 kb long. I have never done such a big piece of DNA
> cloning before. I would like to get help from anyone who has done such or
> who knows any literature (technical) on how to isolate this DNA from virus


This should be a useful reference, by the people who developed the PAC
cloning system:

Ioannou and de Jong, 1996

Construction of bacterial artificial chromosome libraries using the
modified P1 (PAC) system

In Current protocols in human genetics (ed. N.C. Dracopoli et al.), pp
5.15.1 - 5.15.24. John Wiley & Sons, New York

> without break the DNA, and what the best way is to transform the DNA into
> E. coli host (chemical competent cells or electroporation) .

Electroporation, for sure. DH10B is the preferred strain for PAC cloning.

Best wishes

Jose E. Mejia

Wellcome Trust Centre for Human Genetics
Windmill Road, Oxford OX3 7BN, UK
Tel: +44 1865 740021
Fax: +44 1865 742186
Email: jose.mejia at well.ox.ac.uk