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Team:Evry/Protocols/10
From 2013.igem.org
Homologous Recombination
Aim
Preparation
(before going forward all recombination protocols should be done at 30° unless indicated otherwise)Strain Preparation
Transform the PTKred Plasmid with the strain of interest
Start an over night culture with the strain transformed with PTKred at 30°
Dilute the overnight culture in a ratio of 1ml/100ml and add IPTG (final concentration of 2 mM) and Spectinomycin (60mg/ml)
Grow up to OD600: 0.5 then make electrocompetent
Integration:
Prepare the PCR fragment to be integrated flanked by 50 bp genomic sequences
Gel-purify the product and transform with the electrocompetent PTKred strain
Recover in LB for 2hrs with IPTG and spectinomycin
After 2hrs incubation add the selective drug which should be inserted in the genome( ex kanamycin to kan R cassette inserted ) and incubate overnight in liquid culture at 30°
Next day, (the culture should be very turbid or the recombination did not work ) ,plate streak 50 μl of the overnight culture on a plate containing selection drug which in this case is Kanamycin and grow overnight at 30°
Next Day pick a colony and make Glycerol stocks
Elimination of PTKred plasmid ( plasmid Curing):
Inoculate the strain that you have made by Homol.R. in LB overnight with spectinomycin at 30°
Note: adding spectinomycin in this step ensures that the plasmid will not integrate in the genome in a rare occurrence
Dilute 1/100 and grow at 42° for 4hrs and then plate sreak 50 μl of the culture om LB plate at 42°
Next day, Pick colonies and grid plate on LB then Spectinomycin and grow at 37°
Flip Recombinase flip of Anti-biotic Resistance Gene:
Transform a cured strain with PCP20 plasmid
Grow overnight in LB with Cam at 30° overnight
Dilute 1/100 and grow for 3hrs at 37°
Streak plate on LB plates and grow over night at 42°
Repeart the grid plating as in the diagram
