Changing the vector - (Aug/05/2014 )

Crap, yes it appears I put the wrong one in there. Apologies. I'm afraid I'm not perfect...yet.

You may be able to get around this by doing a blunt TOPO cloning and using one of the sites in that vector's MCS with EcoRV. Otherwise you'd need to reorder the reverse primer. Again, my apologies.

No worries! I am learning good things everyday:) May be you can teach me how to ligate blunt PCR product to blunt vector and keep the directionality of the gene ( in correct ordination)?

I am thinking of two ways to do this:

1- to use EcoV for the left side that will give blunt end. and find other blunt side at the right side and do blunt ligation then the directionality problem appears.

2- after ligation reaction that where I am now. gel purify what I think correct size then use it at the transformation step.

What do you think?

You could do that but you'd want to dephosphorylate the vector after digesting it or you'll get a ton of colonies from the vector ligating to itself. After getting colonies, you'd want to do a digest of several clones that will tell you in which direction the onset went. It should be about 50% for each direction.

what about option 2? gel purify the correct size before transformation?

Well it's not a bad idea to gel purify the correct band from your PCR, especially if you see several bands.