Restriction mapping - (Apr/02/2007 )

hello everybody
I did blunt end ligation of 11kb vector and 1.3 kb insert ... after very much struggle i got 3 positive clones..but for confirmation i need to do Restriction mapping but i dont have sequence of gene and so plz tell me how to choose RE...and help me to design this expt..
thanks

santosh

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In allyour expts, I think you need to consider the insert, the plasmid (11 kb) and the construct (12.3 kb). Whatever you do to one, you need to do to the others.

Do you have the map of the plasmid? Find an enzyme that only cuts once and see what you get with unligated plasmid and your construct.
Try digesting your PCR product with a few enzymes and see what you get. Pick a single-cutter that cuts the plasmid only once or twice, and digest the plasmid with it.

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as best as I can see, you have no sequence data on the gene. Thus no restriction mapping. The only way I can see that you can confirm the gene is really present is a test of function.

Is the no data on the gene in any publicly available database?

If you are certain and confident that there is something good in your clones, I would advocate sequecing. If you make primers that sit on your vector that flank your gene, you can sequence the gene. Once you have the sequence. you can compare the 3 genes. You could probably be wary of the odd man out.

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