I cloned an insert to a vector. Then I cloned another sequence (insert 2) to previous cloned vector.
After transformation and performing colony PCR with designed primers for vector backbone, I founded that there are exist two vector simultaneously in all my colony: vector containing insert 1 and vector containing insert 2, simultaneously.
Can I separate this 2 vector with different insert? How?
I dont think you will get the answer since your description is so messy...I do not understand at all what you actually did. Sorry.
Hi Mohammad,
Two plasmids in one cell sounds unusual, unless the plasmids have different origins of replication. There are protein expression systems that use two plasmids in fact, but the plasmids are different.
I see two scenarios:
a. You don't have a single colony, but a mix.
b. Your vector contains a tandem of both inserts. In other words, you don't have two plasmids, but a single plasmids with both inserts.
As you don't provide details of what you cloned an how, it's difficult to tell.
One thing you can do is isolate the plasmid and re-transform, then screen several colonies. You may be able to find colonies with one plasmid each. But if you find that all colonies have both inserts, then scenario b is likely.
Hope that helps.
F
If you construct plasmid harboring insert A (plasmid no.1) and insert B (plasmid no.2) by using the same vector backbone (= same origin of replication). Then, transform two plasmid into same host. Plasmid will soonly loss. You can get more detail in this website. (http://blog.addgene.org/plasmid-101-origin-of-replication)
In my opinion, You can extract plasmid with different insert. Then, checking the size on agarosegel and cut the gel of each plasmid seperately and transform into seperate host.
Dear Di,
You can use same vector by adding some linker between two proteins having pro-tease site. It would be more better than adding two vector in a cell. Particular if the both the proteins will have almost same m.wt.
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