cloning - (Jan/14/2008 )

I am trying to PCR the digested and dephosphorylated insert before ligation to confirm the insert after gel elution.
If i dont get PCR product does that signify any thing else other than absence of insert?????????/

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Failure of the PCR reaction, perhaps might b e easier.
Why not just do an A260 of the eluate? You should only have DNA in the tube, and it will give you the concentration of the insert, which you will need in the ligation reaction.
Why do you want to dephosphorylate the insert? Surely you want the vector treated so you don't select religated single-cut plasmid.

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run the insert on gel to be sure of the presence of an insert.

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