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Need help. Generating mutants by allelic exchange

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#1 cspD



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Posted 29 September 2013 - 11:28 AM

Alright, so I'm generating deletion mutants. I have the my constructed my vector with upstream + downstream regions of the gene I want to delete with a gentamicin resistance cassette fused in the middle. It has a sacB counter-selection marker to select for the true double crossover event. These constructs are in E. coli and I'm doing the deletion in Pseudomonas aeruginosa. 


So everything is working fine. The protocol requires I carry everything out at 30 degress celcius, so everything grows a bit slower and takes longer to grow. I do the conjugation and get gentamicin resistant colonies. I then plate these on sucrose plates and I get sucrose resistant colonies. I thought I had my mutants at this point, but so I isolated these colonies and restreaked them just on gentamicin plates and put them in the 37 degeree incubator so that I could make freezer stocks from them. This is where things go wrong. The very last "step". When I restreak these sucrose resistant colonies and grow them at 37 degrees, they barely grow, or take 2 days to grow. Streaking them on regular LB and putting them in the 37 degree incubator gives good growth, but not on gentamicin which doesn't make sense.


I've been working on this for months now and have had problems every step of the way. Really have no clue what's going on and could really use some suggestions. Thanks. 

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