Transformation of Escherichia coli with large DNA molecules by electroporation

We have examined bacterial eectroporation with a specific interest in the transformation of large DNA, i.e. molecules>100 kb. We have used DNA from bacterial artificial chromosoms (BACs) ranging from 7 to 240 kb, as well as BAC ligaton mixes containing a range of different sized molecules. The efficiency of electro-poration with large DNA is strongly dependent on the strain of Escherlchla coil 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 dramati-cally 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 voitage 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 mol-ecules. 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