Try the cloning reaction with 2:1 ratio of vector and insert. Also, try to increase the incubation time from 5 mins to ~30 mins. Moreover, try to increase (2 to 3 fold) the transformation mix when spreading on the selective plate.
Respected sir the time of incubation you have mention is wht sort of incubation time? Means at wht step?
I mean, after you mix the vector with your gene of interest and salt solution, then usually you will incubate at minimum of 5 mins. If you want to get more colonies, you can increase this incubation time from 5 mins to ~30 mins.
Hi Richa....
I use ligation kit from Genei..... incubation i do frm generally RT for 4 hours and then after that 16 0c for over night.... medium i have tried borth means sumtime i have used LB and some time i have used SOC medium in a quantity of 900 ul per transformation....
Thanks alot Richa i will follow yrs valuable instruction during my next transformation experiment ......
Have you done the appropriate controls? If you transform with your undigested plasmid, you should get hundreds of thousands of colonies. If you don't, then you are having an issue with either your plasmid, the competent cells or your transformation protocol. Ligation times will have some effect on efficiency but given your very low numbers I suspect the problem is not the ligation.
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