Ligation with 3 inserts - (May/18/2007 )
I'm trying to make a longer probe using a ~400 bp insert as a starting point. I cut my vector with XhoI and BspEI to leave the linearized vector and the 400 bp insert, and I want to get 3 inserts ligated head to tail and then to ligate back into the vector. Then I will cut at sites adjacent to the BspEI and XhoI sites to get my ~1200 bp probe.
The probem is I can only ever get one insert ligated.
What I tried so far:
1:50 molar ratio of vector:insert, ~300 ng total DNA
Add insert first, 20 min ligation in 10 ul, add vector, bring up to 12 ul, 20 min ligation.
Also tried the first step with insert only O/N. I used the whole amont for transformation and got ~200 colonies, while a vector only control gave 0 colonies.
I greatly appreciate any help and advice! Thank you!
Since you have already tried concatemerising the inserts together by a overnight ligation with little sucess, I would recommend redesigning the insert primers. There are better ways of doing this.
However if a primer redesign is not possible I would recommend trying quick ligase on your inserts (or adding 15% PEG6000 final) A ligation time course on the quick ligase might be useful.