Non-directional cloning - (Apr/10/2006 )
how likely is it that all your clones contain the insert in the wrong direction after non-directional cloning? What could be reasons for that? I cut both the vector and gene with EcoRI, treated the vector with Antarctic Phosphatase and ligated them.
I checked the clones by restriction digestion but was getting strange results (some positive, some negative).
If I get what you're doing, you should theoretically get 50% in the right direction and 50% in the other direction.
Maybe you should go into more detail about "strange results" (i.e.: mostly wrong direction, empty vector, other things...). Or maybe go into more detail about you're entire procedure (meaning: do you PCR your gene of intrest or cut it out of a vector and so on).
Right, I cut the gene out of pGEmT easy with EcoRI and ligate it into pcDNA3.1+.
When doing the restriction digestion of the plasmids with different enzymes I get mixed results in terms of whether I have the right orientation of insert or not, depending on which enzyme I use.
90% of the clones I checked (20 so far) contained an insert. Some were cut quite strangely though (sizes of bands were not what I expected but the total size was ok). I was thinking that I maybe created new restriction sites during cloning. But I also think that this is very unlikely because non-directional cloning is pretty simple as you only use one enzyme.
I expected a 50-50 chance of getting the right direction of insert. Could this only be bad luck?
Is it possilble that your pGem T easy prep is contaminated with another plasmid? It doesnt sound like concatomers, but even if it was, are you sure you would see the difference in size in the whole plasmid? I would digest your original plasmid prep with a couple of different enzymes unless this same prep was sequenced with no problem or you have another good reason to believe it is completely pure... Did you get rid of the T-easy somehow to be sure it is not vector contamination from T-easy ligating with the 3.1?
It was indeed kind of tricky to seperate the gene (2,7 kb) from pGemT (3kb), so I am not completely sure that I got 100 % rid of it. But I checked the restr. dig. results and they don`t match with a possible pGemT-pcDNA3.1-construct either. I sent 3 different plasmids for sequencing to figure out what they consist of.
I think I have to check more clones. Does anyone have a protocol for isolating plasmids without using a kit and columns? Could become quite expensive otherwise.
You could be selecting against the "correct" orientation due to expressing a toxic gene product.