This phenomenon is sometimes seen if all of the transformation mix has been plated out or all of the ligation mix was used in the transformation. It is indicative of a failed ligation experiment. The blue colonies are the undigested vector molecules and the odd whites are simply due to mutant undigested molecules with non functional alpha peptides. These molecules will always be present to a very low extent in all populations and are rarely seen when plating out the volumes detailed in the technical manual. Tips to boost the number of colonies obtained.
Higher temperatures tend to favour blunt end ligations.
This ensures that the cells are subjected to the shock temperature for the necessary period of time with no significant lag period for equilibration. Increase the time of the heatshock from 45 to 90 seconds. Incubate this reaction at 72 C for 3 h, clean up the sample as normal and proceed with the pGEM -T protocol. References Storts, D.R. and Mezei, L.M., Cloning PCR Products, in PCR Technology, Current Innovations, Griffin, H.G and Griffin, A.M.,Eds., CRC Press. Sambrook, J., E.F. Fritsch, and T., Maniatis.