I have an insert and vector with one sticky end and other blunt end, It did work when insert size was small but at the time of large size fragment it didn't. Can anybody provide me solution?
You could use a polymerase to fill your sticky ends and do a blunt end ligation. The Klenow fragment (part of polymerase I) should do the job.
There are many advices for ligations around. Using a low temperature and incubation for a long time (e.g. 4 C over night) could be helpful. Moreover, try different ratios of insert:backbone molecules starting from 3:1.
Thank You Abhishek and boas. When I did ligation and confirmed it by digestion all were insert negative, I will try both ways that you have mentioned. hope it will work. I am also planning to give alkaline phosphatase treatment to reduces the chances of self ligation. hope it will work... Thanks