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Làm sao biết đoạn insert đã gắn vào trong plasmid (sau khi ligation).

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Hỏi: How can I confirm an insert in a plasmid without sequencing?I am trying to clone a gene from E.Coli and after the PCR I got nice band. I cut the band and purified the DNA and ligated it to a vector. After the ligation I checked it in the gel whether I got the insert. My question is, my bands look same after the PCR and the ligation, so how can I be sure that the gel run after the ligation has the insert if I won't do the sequencing?


  • I assume that you cloned your gene via restriction sites. If so, then you can cleave your ligated vector with the respective enzymes and release your insert. This insert should have the correct size. Additionally, you can cleave with two restriction enzymes, one of which cuts only in the vector and one of which cuts only in the insert. This way you can check whether you actually have an insert and you get an idea whether it is the right thing. In any case, if your digests look promising you will absolutely have to sequence your insert, even if you are dead sure that you cloned the correct fragment. This is because by the PCR that you employed to amplify the gene from E. coli might have introduced mutations (even polymerases such as Pfu produce errors every once in a while). You do not want to find out about that after you worked on the construct for a few years ... ;-)




(Nguồn: ResearchGate.Net)

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1) do a colony PCR with a forward primer for the vector and reverse for insert . 
2) do a digestion of your clone and empty vector and run them on a gel . ideally your clone should give a pop out of your insert 
3) if your insert is of significant size say above 500kbp, running both the empty vector and the clone on a gel against a ladder , you should be able to see the difference.  
i would still recommend sequencing just to make sure there is no mutation. If you are amplifying with Taq  then there are chaces of mutations because Taq has no proofreading capabilities. You should then go for a high fidelity enzyme . 

How can I confirm an insert in a plasmid without sequencing? - ResearchGate. Available from: https://www.researchgate.net/post/How_can_I_confirm_an_insert_in_a_plasmid_without_sequencing [accessed Dec 1, 2015].

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Try performing some restriction digestion using enzymes which were used for cloning into plasmid or if you know the gene sequencing use few sets of  restriction enzymes to digest the gene and look for specific size bands on the agarose gel.
How can I confirm an insert in a plasmid without sequencing? - ResearchGate. Available from: https://www.researchgate.net/post/How_can_I_confirm_an_insert_in_a_plasmid_without_sequencing [accessed Dec 1, 2015].

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Personally I would prefer to do a colony PCR at first. Additionally you can check correct insertion by restriction endonucleases. Just look for nice restriction sites which gves you one or more nice visible fragments in gel. Nevertheless I would also sequence the gene if it should be used for downstream analysis. Because it can always happen that a polymerase error occured in amplification process - even with a prrofreading polymerase. Good luck!

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For me the best way to quickly check positive transformants is to design an insert specific primer + a vector specific primer and make colony PCR. Understandably I never have positive control plasmid, but I always have some new positive clones which give PCR product of proper length :-) But  I alwaysseque nce my positive clones because of mistakes which can be made by polymerase during amplification of the  insert. Good luck!!!

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