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?
Hello Kailash,You can use PEG 4000 in your ligation mixture as one of your end is blunt. It has worked for me, hope it will work for you as well.
Hi Kailash,
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.
Good luck!
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
You can try to do the ligation overnight at 16C. Low temperatures helped the ligation of long inserts.
Good luck
You could try Seamless In-Fusion Cloning.I ligated 8kb insert with 11kb vector by Seamless In-Fusion Cloning.
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