urgent help regarding wanner method trouble shooting - (Jun/26/2009 )

Hi all,
I would like to make a mutant of gene in my Aeromonas bacteria.And I am using PCR based inactivation of wanner protocol.Essentially i did as following,
1,Made a competent cells of wild type bacteria
2.Transformed with a pkd 46 plasmid which produces red recombinase.Prepared competent cells.
3.Primers were designed with a homology to my gene and pkd3 plasmid.Did PCR and purified and DpnI treated and ethanol precipitated.And concentration was 85ng/uL in total 7uL.
4.Bacteria was electroporated with this DNA and grown in SOC media and plated on chloromphenecol 5ug plates and grown at 37C
5.I have repeated this protocol more than 10 times changing different parameters in it.Even then no luck .Any help in this would be highly appreciated.Eagerly waiting for reply .

sunni on Jun 26 2009, 12:34 PM said:

Hi all,
I would like to make a mutant of gene in my Aeromonas bacteria.And I am using PCR based inactivation of wanner protocol.Essentially i did as following,
1,Made a competent cells of wild type bacteria
2.Transformed with a pkd 46 plasmid which produces red recombinase.Prepared competent cells.
3.Primers were designed with a homology to my gene and pkd3 plasmid.Did PCR and purified and DpnI treated and ethanol precipitated.And concentration was 85ng/uL in total 7uL.
4.Bacteria was electroporated with this DNA and grown in SOC media and plated on chloromphenecol 5ug plates and grown at 37C
5.I have repeated this protocol more than 10 times changing different parameters in it.Even then no luck .Any help in this would be highly appreciated.Eagerly waiting for reply .

What temp did you grow your pKD46 plasmid containing bacteria at before you made competent cells?? pKD46 is temp sensitive so you should be growing at 30C.
Could this be your problem??