Plasmid purificaction

[Chris Boyd]

JuliaRaquel Acero Reyes ICP IDA ECP (jacero at INFANTAS.ECP.COM) wrote:

: I need a protocol for the isolation of big plasmids (80Kb). I will 
: appreciate any kind of help.

The most consistent method in our hands is Qiagen Midi column
purification.  A modification of their standard protocol for P1 clones
(similar size to PACs) is in Qiagen News Issue 1/95. Alternatively,
traditional alkaline lysis followed by CsCl/EtBr gradients also works.
Don't expect either method to give you anything like the yield of
plasmids such as pUC18 etc.  About 100ug per litre of starting
culture is standard.

See also the important paper,

  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