How long is too long for a TA vector? - (Nov/21/2006 )

Hello,

I just realized I'm using Promega's pGEM-T vector which is 3kb, but the full insert I want to put into it is 6kb. The insert is twice the size of the plasmid. Is this going to be a problem?

Also the GFP plasmid I'm going to eventually put said insert into is again only about 1/2 the size of the gene I'm looking at. When it comes time to clone into this plasmid, would it be prefferable to dephosphorylate the insert or the plasmid?

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Its always wiser to dephosphorylate the plasmid because it contains the origin of replication. Circularisation of vector will replicate in your transformed cells resulting in high background whereas, cirucularization of your insert will not replicate hence no background. Although with pGEM-T (as you already know), you don't need to dephosphorylate since it cannot religate to itself. Dephosphorylating the insert may improve your ligation (by preventing insert circularisation and insert concatemers) although I haven't tried it before and I'd be curious to hear if others have used this approach with pGEM-T.

There shouldn't be a problem with ligating large fragments into pGEM-T although sometimes large inserts can affect copy number resulting in less DNA when you purify the vector. You just need to do a maxi rather than a mini prep. It will be less efficient so ligate overnight rather than the 1 hour that the kit recommends.

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thank you very much

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