boyana

Hi! I am currently trying to perform an LR recombination reaction between the positive control of the LR reaction kit pEntr-Gus(donor vector) and the entry vector pDest32. I am trying to perform this reaction successfully and then try to do it with the actual cDNA library, which is already put in a donor vector. For some reason I cannot make my positive control work. I believe that it is a problem with the LR reaction, and not the transformation of the reaction into DH10B cells, because my transformation of the cells with the positive control pUC 19 works perfectly. It is just that they cannot be transformed by the DNA in my LR reaction. Could it be that all the cells are killed during the electroporation process because of high salt content in the LR reaction( I do an ethanol precipitation before I put it into the DH10B cells). What is the most effective way to get rid of the salt? Could it be another problem with the LR reaction, that I just can't put my finger on? I have been trying to do this several months now. Please help. Thanks to everybody.