2 replies to this topic

[elk2019]

When I ligate my insert to my vector, perform my transformation, and plate my cells, I get plenty of colonies, and my colony PCR indicates that my ligation reaction worked. For my colony PCR, I include a positive and negative control, and my forward primer is in the insert and my reverse primer is in the vector. When I go to miniprep my colonies however, I have very low DNA yields (~20ng/uL). I have sent this for sequencing anyway, and found that the sequence is garbage. Prior to miniprep, the LB solution is cloudy suggesting I am getting ample colony propagation. I have tried changing my LB Broth with ampicillin (operating under the assumption that the ampicillin might be degrading and allowing for competitive growth from other bacteria). I have also tried switching miniprep kits, and still the same results. I have also tried using different competent cells for the transformation. My insert is 2.5 kb and my backbone is also 2.5 kb, seems like a very manageable sized vector. I am very confused as to what might be happening here. I would appreciate any suggestions you might have!

Edited by elk2019, 19 May 2012 - 12:18 PM.

[phage434]

I would try the pcr on the miniprep sample.  It is possible you are pcr'ing from ligated DNA on the plate, rather than from plasmid from the colony.  Have you run the miniprep sample on a gel?

[elk2019]

You were absolutely right. I was getting positive PCR results from the ligated DNA on the plate. My LB agar plates were old, the ampicillin was degraded, and I was getting competitive growth from bacteria inside the incubator. I made new plates with carbenicillin this time, and all went well. Thanks for your help!