Nucleic Acids Research, 1995, Vol. 23, No. 11 1990-1996
© 1995
MOLECULAR BIOLOGY |
Transformation of Escherichia coli with large DNA molecules by electroporation
Division of Biology 147-75, California Institute of Technology Pasadena, CA 91125, USA
* To whom correspondence should be addressed at present address: Whitehead Institute/MIT Center for Genome Research, 1 Kendall Square, Building 300, Cambridge, MA 02139, USA
Received January 24, 1995. Revised April 4, 1995. Accepted April 4, 1995.
We have examined bacterial electroporation with a specific Interest in the transformation of large DNA, I.e. molecules >100 kb. We have used DNA from bacterial artificial chromosomes (BACs) ranging from 7 to 240 kb, as well as BAC ligatlon mixes containing a range of different sized molecules. The efficiency of electroporation with large DNA is strongly dependent on the strain of Escherichia coll used; strains which offer comparable efficiencies for 7 kb molecules differ In their uptake of 240 kb DNA by as much as 30-fold. Even with a host strain that transforms relatively well with large DNA, transformation efficiency drops dramatically with Increasing size of the DNA. Molecules of 240 kb transform ~30-fold less well, on a molar basis, than molecules of 80 kb. Maximum transformation of large DNA occurs with different voltage gradients and with different time constants than are optimal for smaller DNA. This provides the opportunity to Increase the yield of transformants which have taken up large DNA relative to the number incorporating smaller molecules. We have demonstrated that conditions may be selected which increase the average size of BAC clones generated by electroporation and compare the overall efficiency of each of the conditions tested.
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