TRANSFORMATION OF ESCHERICHIA-COLI WITH LARGE DNA-MOLECULES BY ELECTROPORATION

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 24 0 kb, as well as BAC ligation mixes containing a range of different si zed molecules, The efficiency of electroporation with large DNA is str ongly dependent on the strain of Escherichia coli used; strains which offer comparable efficiencies for 7 kb molecules differ in their uptak e of 240 kb DNA by as much as 30-fold, Even with a host strain that tr ansforms relatively well with large DNA, transformation efficiency dro ps dramatically with increasing size of the DNA. Molecules of 240 kb t ransform similar to 30-fold less well, on a molar basis, than molecule s of 80 kb, Maximum transformation of large DNA occurs with different voltage gradients and with different time constants than are optimal f or smaller DNA, This provides the opportunity to increase the yield of transformants which have taken up large DNA relative to the number in corporating smaller molecules, We have demonstrated that conditions ma y be selected which increase the average size of BAC clones generated by electroporation and compare the overall efficiency of each of the c onditions tested.