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
