Hello,
I am facing a problem in clonning of 45 bp insert in 3.5 kb plasmid. Actually i am getting self ligation of vector rather than with insert ligation. I run a control (only vector) parallel with the ligation of insert and the result with control is 2-3 colonies while with insert 10-12 colonies. From these 10-12 colonies only three are clonned with insert.
I have gone through following reasons.
1. Problem with dephosphorylation of vector (i did it again)
2. I varied the concentration of insert.
3. I varied the concentration of vector.
4. I have checked the several combinations of insert and vector ratio (1:3 to 3:1).
5. Vector concentration is 10-15 ng in per ligation reaction.
The main problem is that i am getting more colonies with self ligated vector in the plate where insert was supposed to ligate while the control where no insert was present i usually get 2-3 colonies. I know that it is normal because covalent linkage is made within the bacetria by DNA repair mechanisms.
Milki
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