Large DNA cloning?

[Chris Boyd]

Jose E. Mejia (jose.mejia at well.ox.ac.uk) wrote:
: 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.

Be sure also to study,

  author = "Sheng, Y. and Mancino, V. and Birren, B.",
  title = "{Transformation of {\it Escherichia coli} with large DNA molecules by
electroporation}",
  journal = "Nucleic Acids Research",
  year = "1995",
  volume = "23",
  pages = "1990-1996",

Best wishes
--
Chris Boyd                       | from, | MRC Human Genetics Unit
chrisb at hgu.mrc.ac.uk             |  not  |  Western General Hospital
http://www.hgu.mrc.ac.uk/~chrisb |   for |   Edinburgh EH4 2XU, SCOTLAND