We are using the reporter construct pPelican to identify the regulatory region of the Agr gene. We are able to purify the reporter using the Qiagen mini-prep kit, but when we use the midi-prep kit, we cannot isolate pPelican DNA. The midi-prep kit works fine with other plasmid constructs. pPelican DNA purified with the mini-prep kit is the right size (around 13 kb) and seems to contain the correct restriction sites for this vector.
We tried ligating small DNA fragments (240, 640, 1600 bp)into pPelican and transforming DH5-alpha cells with the constructs. We get transformed colonies on an agar plate with antibiotic. We streaked the colonies on a second agar plate with antibiotic. The colonies containing the construct with the largest insert didn't grow, but those colonies containing the smaller inserts did grow on the plate. When we try to grow the colonies from the plate in L broth, nothing grows. We don't get growth if we include antibiotic, and we don't get growth if we leave out the antibiotic.
Does anybody have any suggestions concerning what the problem might be and how we can get transformed cells to grow in L broth or some other liquid medium so that we can purify and characterize our construct?
Associate Professor, Biology
University of Texas of the Permian Basin