I am trying to clone my gene of interest in pCAMBIA2310 vector by excising out the GUS site with AhdI and DraIII double restriction digestion . Gene of interest with specific restriction site i.e AhdI and DraIII was purified. pCAMBIA 2301 vector after double restriction digestion produces 2 bands one band is of gus gene and other one is vector backbone, vector backbone was purified. Both purified gene of interest and pCAMBIA 2301 vector backbone was ligated and after that transformation experiment was performed but after transformation i did'nt get colonies on my kanamycin LB agar plate . Has anyone faced similar problem ? Any suggestion would be greatly appreciated .
consider to have 100microgram/mL kanamycin antibiotics in your LB, then you may look back at transformation, like give it a try on ice for 30 mints, then for 45 sec at 42 degree Celsius in water bath, then 3 mints on ice and finally add one mL LB without kanamycin to the suspension, incubate at 37 degree Celsius at 220RPM for one hour and then plate for overnight. moreover consider your DNA is between 50-100ng/microliter. best of luck
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