PCR cloning: neg self-ligation control, many colonies but no insert - (Jul/23/2007 )
Hi
I ve troubles with my pcr cloning. I have a 600 bp PCR insert I want to ligate in the pQE30 plasmid. I restrict the vector and the insert with HindIII and BamHI (I did also cross digestion with the vector and both enzymes worked) and purified them with qiagen pcr purification kit. Ligation was over night at 14°C in a molecular ratio of 1:5. When I did the self-ligation test I had only one colony. Transformation with the insert yielded in 100-200 colonies, but when I extracted the plasmid and digested it, it had no insert??? I also run the empty vector and the ligated vector on a gel, and they have the same size, so it s not a problem of digesting out the insert, its just not there.
So why do I have no insert when the self-ligation was negative?
I also have 3 aditional bp for my primer restriction site to make sure that the enymes can cut.
I would be really glad, if any of you had an idea to solve this!! Thanks!
I ve troubles with my pcr cloning. I have a 600 bp PCR insert I want to ligate in the pQE30 plasmid. I restrict the vector and the insert with HindIII and BamHI (I did also cross digestion with the vector and both enzymes worked) and purified them with qiagen pcr purification kit. Ligation was over night at 14°C in a molecular ratio of 1:5. When I did the self-ligation test I had only one colony. Transformation with the insert yielded in 100-200 colonies, but when I extracted the plasmid and digested it, it had no insert??? I also run the empty vector and the ligated vector on a gel, and they have the same size, so it s not a problem of digesting out the insert, its just not there.
So why do I have no insert when the self-ligation was negative?
I also have 3 aditional bp for my primer restriction site to make sure that the enymes can cut.
I would be really glad, if any of you had an idea to solve this!! Thanks!
hi
are you doing this cloning for the first time or have you done this before? i don't know much about pQE30 plasmid, is it an expression vector?
How big is the vector? And in your self ligation test, did you add ligase or not?
My vector is 3.4 kb and for the self ligation I used the same conditions as for the normal ligation (just without the insert).