Ligation at 16°C,ON: where to keep it till transformation? - (Feb/05/2007 )
I had problems in my cloning and then some people suggested me to do it overnight at 16°C.
Since it takes 14 hours till you see the colonies after plating, I would like to plate in the evening. My question is where should I keep my product till I transform it?
Thanks for your replies.
you may freeze it or keep it at 4 Celsius.
Though, I would simply transfrom the cells, and plate. But rather then immediately incubate the plates. I leave the plates at room temperature. And when evening comes, only then do I incubate the plates at 37 Celsius
Hi, You can either go on with the ligation and keep the tube at 16°C, or keep the tubes on ice (other technique for ligation is overweekend at 4°C) or transform and after the incubation in SOC medium you can keep the tube on ice, or plate directly and leave the plate at RT until the evening comes. Don't worry, if the T is not higher of 20°C either bacteria or DNA are not affected. Good Luck!
Thank you! So I transformed and plated and kept it at room T till the evening and when I am about to leave I will put them to incubator 37°C.
I hope I get some positive colonies.
I leave my ligations on the bench at RT and use them later to get the same results.