homologous sequense 16nt long works. I've never tried shorter . Did you try betaine or formamide to reduce Tm?

I didn't try using betaine or formamide, but I think that maybe is cheaper to buy new primers than by that compounds.

Thanks for your answer!!

I have tried till 15 and it worked for me.

Thanks for your answers

I'm Finally going to use 2 pairs of primers, one without connectors, to amplify my sequence in the cDNA, and one with connectors to use later for cloning.

I think I'll try first cloning directly with the PCR made with conectors, and if that doesn't work, I'll use T/A plasmid cloning to be sure that the PCR is working.

Thanks

Hello, David.

Don't know, whether you've already solve your problem. If not, I reviewed my cloning schemes and find primer for step- out pcr (with RE target and some axtra sequence, Tm=73) with homologous sequence 13 nt long. I don't think such short homologous sequence will be good if matrix for you pcr is genomic DNA. But if matrix is some plasmid (in my case it was) such primer works. I used Tm of homologous part of primer for 2 cycles (time of hybridization was about 40 seconds) and Tm=67C for remainder 22 cycles (time of hybridization 20 seconds).

Thanks for your coment Natalia, Unfortunately I'm working with cDNA from cells because my secuences its a splicing variation that no one cloned before...