gene deletion via the RED recombinase method - are you experienced? (May/24/2005 )

my lab is using the RED recombinase method [essentially based upon the protocol of Datsenko and Wanner 2000] to make gene deletions in various strains of E. coli and Salmonella.

[Briefly, the initial steps involve the transformation via electroporation of cells [from a restriction negative strain] containing a low-copy number plasmid inducibly expressing the phage lambda RED recombinase genes, with a selection determinant [ie Kanamycin resistance] flanked by 42bp homologous to selected regions upstream and downstream of the gene to be knocked out, brief incubation [and frantic finger-crossing] for recombination to occur, and selection plating.]

Despite some modification of the protocol for our strains, our successes in knocking out targeted genes have been few and far between. A recent example - three people between us performing up to 20 different transformation assays per day have taken about 3 weeks to putitavely knock out two genes.

Perplexingly, we are very occasionally blessed with many transformant colonies all at once. This implies to us that there must be some sort of secret involved in this technique we don't know about which increases the efficiency. We are also having great difficulty replicating past successful knockouts [although this is of secondary importance]. there's got to be something we're missing!

If anyone's had experience in this technique, I'd be really appreciative of ANY tips, hints, strange rituals or lucky underwear stories that may have enhanced the efficiency of the gene knockouts for you.

Cheers!

my lab is using the RED recombinase method [essentially based upon the protocol of Datsenko and Wanner 2000] to make gene deletions in various strains of E. coli and Salmonella. 

[Briefly, the initial steps involve the transformation via electroporation of cells [from a restriction negative strain]  containing a low-copy number plasmid inducibly expressing the phage lambda RED recombinase genes, with a selection determinant [ie Kanamycin resistance] flanked by 42bp homologous to selected regions upstream and downstream of the gene to be knocked out, brief incubation [and frantic finger-crossing] for recombination to occur, and selection plating.]

Despite some modification of the protocol for our strains, our successes in knocking out targeted genes have been few and far between. A recent example - three people between us performing  up to 20 different transformation assays per day have taken about 3 weeks to putitavely knock out two genes.

Perplexingly, we are very occasionally blessed with many transformant colonies all at once.  This implies to us that there must be some sort of secret involved in this technique we don't know about which increases the efficiency.  We are also having great difficulty replicating past successful knockouts [although this is of secondary importance]. there's got to be something we're missing!

If anyone's had experience in this technique, I'd be really appreciative of ANY tips, hints, strange rituals or lucky underwear stories that may have enhanced the efficiency of the gene knockouts for you.

Cheers! 

hansel,

thank you very much for your help. the links were really useful, and we're now considering using longer homology regions for our more recalcitrant salmonella knockouts.

[if it helps anyone, we've also found growth in 2YT and arabinose induction for 2 rather than 4 hours prior to electroporation seems to increase efficiency, although, as mentioned in the kim protocol, this is just a vague notion.]

cheers!

I know it past almost one year from your first message, but still, I hope you are still working on it. I'm also trying to knock out gene from Salmonella and I'm having hard time. Were you successful in knocking out gene? What are the different things you've been doing from the original protocol? I really appreciate any help.

Atska