problem with generation of restriction digestion site - (Jun/29/2005 )

hi

I did generate a restriction site for hindIII in my clone by using PCR . the sequence of HindIII is created in the primers.

the PCR product has the correct size, I did clone it in pUC18 and transform E Coli, everything seems to be ok , but when I extract the recombinant plasmid and try to cut it with HindIII, the digestion is not succesful?
Does anyone has any suggestion on what is going on?
is it possible to have a mutation in the restriction site even if I did run my PCR with a high fidelity enzyme from Roche??

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Double check your primer sequence (the sequence you generated originally AND the sequence on the primer documentation).

Double check your restriction conditions.

Triple check the primer sequence

Try sequencing the region of the plasmid with the restriction site in it.

Quadruple check your primer sequence

you could also put jsut your primer sequence into a dna sequence analysis program and run a restriction enzyme check to make sure the HindIII site is correct

It is also possible that your primers are dodgy (this is rare but it does happen).

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Are you sure that you have your insert cloned in the vector in the first place. Cut your plasmid with a different enzyme (cut in vector only and not in insert) to check the linear size of your plasmid. It should be euqal to vector size + insert size. If not then you might not have cloned in your insert properly.

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hi
have you sequenced your plasmid? I've got cases in which i succeded in cloning my fragment, but one restriction site was abolished by a mutation...

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You could also be seeing methylation of your sequence, depending on the strain of coli you are cloning in. HindIII is sensitive to methylation of the outer A's and inner C's. See http://rebase.neb.com

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