klas@dick.imb.uh.edu (Klas Hedenstierna) (03/06/91)
Recently, I ran into some unexpected problems with a simple transformation experiment. I prepared frozen competent cells of JM109 according to Sambrook et al. (Sambrook, J., Fritch, E. F., and Maniatis, T. Molecular Cloning, second edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor. 1989; originally from Hanahan, D. 1983. J. Mol. Biol. 166:557), transformed 100 microl cells with 10 microl ligation reaction, and got no transformants at all (a +control showed that the competency was reasonably high; between 10^6 and 10^7; selection was for ampicillin resistance). I repeated the ligation, which was a straight forward ligation of an approximately 2 kb fragment into pZ152 (a pBR322 derivative phagemid carrying the M13 IG region), this time checking that the ligation was working. Both the vector fragment and the insert were extracted from an agarose gel with DE81 paper, eluted from the paper with a buffer containing 1.5 M NaCl, EtOH precipitated, and washed with 70 % EtOH. My ligation buffer (10x) contains 250mM Tris, 50mM MgCl2, 50mM DTT, pH 7.6. In the transformation I used less of the ligation reaction than the first time; 5 microl per 100 microl cells. In addition to the normal +control (2 ng pUC18), I also included a control where I added both 2 ng pUC18 and 5 microl ligation reaction, and this plate gave only around 10 % as many transformants as the normal +control. The ligation reaction must therefore contain something that inhibits the transformation. Does anyone have any ideas as to what the inhibitor might be? The second time I got some of the transformants I was looking for (I think), so my immediate problems are over. It would however, be helpful to know what is causing the decreased tranformation efficiency. Many lab manuals mention that the volume of DNA added shouldn't exceed 10 % of the volume of competent cells, but I haven't found any proposals about the nature of the inhibitor. It is of course possible to clean up the DNA in various ways, but I have used the ligation reactions directly before without problems, and, as this is rather convenient, it would be nice to be able to continue doing so. The only potential source of the problem I can think of myself is that I didn't do a phenol extraction after eluting the DNA from the DE81 paper. The original protocol does contain such an extraction step, but I have omitted it before without encountering this problem. All suggestions or guesses will be most welcome. Klas O. F. Hedenstierna University of Houston Department of Biochemical and Biophysical Sciences klas@dick.imb.uh.edu