Lactococcus workers! - (Sep/22/2008 )
Dear all,
I have tried cloning in Lactococcus for the past 3 months. I have checked DNA concentrations for digestion (increased and decreased), played with different ligation compositions, transformed up to 20ul of ligation product into my competent cells and even cloned my insert into pGEM-T easy vector and verified the RE sites of my primers. I am using Fermentas REs-tried both conventional and Fast buffers.
I have tired everything and up till now, no colonies have been obtained, also confirming that my vector has been properly double digested. I have included + and -ve controls for my transformation and my +ve control produces lots of colonies, confirming that my cells are sufficiently competent.
I am at my wits end, and am now preparing ligation in large scale and purifying it before transformation. I don't know if this is a crucial step, btu am trying it out.
I wonder if anyone out there has pointers/hints/advice for me?
Help appreciated!!
Maybe the gene you are trying to transform into your bacteria is toxic.
Can you transform your Lactococcus with another plasmid? What plasmid are you attempting to transform with?
im cloning into pNZ8048. I have tried with pMG36e before and it works ok. Cloning is the only problem....
and the strain I am using is L. lactis MG1363 Are you attempting to ligate and transform directly into Lactococcus? If so, your transformation efficiency may be too low for this to work effectively. In general, it is far easier to first clone into a shuttle vector in E. coli, miniprep the DNA, then transform the miniprep DNA into the Lactococcus. The ability to transform with prepared plasmid DNA does NOT guarantee that your cells are sufficiently competent to directly transform with a ligated product.