in coloning , we need to add junk seq, kozak seq and restriction enzyme seq to the primers but in conventional PCR we do not need to add anything

Hello,

You have to have the sequences of the used restriction enzymes in cloning primers, while you do not need to do anything with regular primers but getting the sequence of your targeted gene taking into account the criteria required for primer design.

Good luck

You need to design primers that will amplify the entire gene, not just a region. Are you trying to clone the genomic DNA (with introns, promotor, etc.) OR are you making a protein expression vector from cDNA (exon only)? Write out out cloning scheme and have someone double-check it before you start.

Also, buy a TA-topo cloning kit. Use a DNA polymerase that gives an A overhang on your PCR product and you can directly clone your PCR product into the plasmid. No need to mess around with adding restriction sites, phosphatase treatment or any of that nonsense.

Thank you everyone for your input.

Katie A Clark

I am going for cDNA, thanks once again for your detailed answer.