Generation/Transformation into DY380 cells - (Nov/19/2005 )
Hi, Just looking for some troubleshooting advice on generating electrocompetent DY380 cells and transforming a PCR product into these cells.
I am currently growing up cells in 1:50 dilution to OD600 of 0.4-0.6. Inducing 10mls of culture at 42C with shaking (8-10min). Cooling in ice slurry for 10 ml then pelleting and resuspending into 100ul of H20. I use 50 ul for each transformation with 200ng of PCR product by electroporation at 2.5-kV, 25-uF, and 200 ohms in 0.2cm cuvetter (Biorad machine). Then incubating in LB for 1 hr before plating on LB-Agar. (protocol based on Yu, 2000)
After all this, I get no colonies!!!!!!!!!! Any ideas, or suggesting? KW1 
I might be missing something here, but PCR products are typically DNA fragments...and do you have some sort of selection in your LB-agar?
if you transform cells with a fragment and not a plasmid or other self-replication piece of DNA, and expect it to grow on antibiotics, it is no wonder you are not seeing colonies
am I misunderstanding what you are doing?
if you transform cells with a fragment and not a plasmid or other self-replication piece of DNA, and expect it to grow on antibiotics, it is no wonder you are not seeing colonies
am I misunderstanding what you are doing?
Sorry...I guess should have included more information. The DY380 bacterial cells already contain BAC DNA (with its own antibiotic resistance to Cm) and I am transforming in a PCR product containing another antibotic gene (Kan) with overhangs that should recombine with the BAC DNA.
I have appropiate antibotic selection for the both Cm in the BAC and Kan in the PCR product (in my LB-Agar).
To clarify for others on the board: DY380 is a strain which is designed to have a temperature inducible lambda RED recombination system. The poster is trying to recombine a PCR fragment into a BAC plasmid contained in this cell by PCR of a product with primers 5' homologous to the insertion site, induction of the DY380 lambda RED system, and electroporation of the PCR product.
The protocol looks ok, but I would be sure to keep the cells cold during the centrifugation and electroporation process. This protocol cries out for some good positive controls. If you don't have them, it is time to make them. I might suggest one which recombines into the genome rather than into the BAC.
Hi, does anyone have any suggestion for the recombineering experiment? I`m trying to recombine a PCR (DpnI digested and gel purified )or a linearized vector (previously dephoshorilated and gel purified) into a strain containing a BAC. But I have colonies with just a re-ligated plasmid). I still have colonies in my non induced plate, but much less than induced one. Any suggestion? Thanks