I have a question about a phenomenon in my ampisillin agar plate.
I've tried cloning 3,8kb PCR product to KpnI/BglII of the 7,5kb vector. This is a Kanamysin resistant vector. Despite several attemption I can't obtain any colonies after transformation into DH5a-cells. The kanamysin agar plates are clear. I have positive control (the intact vector) in transformation, which is ok. (I've asked this in topic cloning long fragments)
I've also tried another plasmid, which had KpnI/BglII sitse (pBLCAT2). I've digested it and gelpurified the bands 2,8kb and 1,7kb, and religated this. This is a ampisillin resistant vector. I can't get a single colony from this control ligation step either, but what I see in these ampisillin plates is a kind of mat surface. What is it?
Once, I also tried to religate HindIII cut a pcDNA3 plasmid and it gave me a same phenomenon. I took a swipe of this mat to new plate, but nothing grew there.
The only time i got some colonies is the time I didn't do gel purifying step to my fragments. (And I assume it all was backround, as I tested few colonies).
Any hints to my clonig problem are also welcome (I have tried different ligation temperatures, times, DNA amounts in transformation etc...)
Thank you in advance
Edited by Heini, 22 February 2005 - 05:39 AM.