Primers with RE sites - (Jan/16/2007 )

I have to design primer pairs with RE sites for the target gene amplification from genomic DNA of Bdellovibrio bacteriovorus 109J using PCR.
Previously I designed the primers with the RE sites on the 3' end since I wanted only the target gene to be amplified. They did not work. I got very distinct bands on my gel for 3 of my genes that were 50bp in size when the size of my genes vary from 600-3000 bp.
I recently read that the RE sites have to be inserted into the 5' end.
Nde1 RE site is the only one that can be incroporated into 2 of my target genes and BamH1 site in the other 2 genes.
Nde1 has an ATG (Start Codon) incorporated within which I would like to exploit but dont know how.
How should I effectively design the primer such that I get to amplify just my gene which would be cloned into pET vector for expression in E.coli.?
Should I check the melting temperature of the primer without including the RE site or is the melting temperature of the whole primer valid?

Your enzyme sites need to be at the 5' end of you primers for both forward and reverse (if your are putting enzyme sites at both ends). If you intend to clone the PCR product directly into the pET vector, you need to add several bases 5' of the enyzme sequence in order to facilitate effiecient recognition of the RE with the recognition sequence. Otherwise you can clone the PCR product into a TA clonign vector and cut out of that. A start codon in the enzyme sequence can be utilized if it is inframe with your coding sequence. Sometimes you can add bases upstream so that it is in a good Kozak sequence.

Should I check the melting temperature of the primer without including the RE site or is the melting temperature of the whole primer valid?

The Tm is only calculated for the hybridizing part of your primer, i.e. that part that binds to the target sequence. Thus, you would not include the enzyme bases as part of your Tm measurement.