ribosomal binding for pcr product cloning - urgent!!! (May/01/2006 )
Hi all, i need to prepare primers for a pcr product cloning, my product needs to have a restriction site and a ribosomal binding site, so i need to add these sequence to the primers but i don't know if it is necesary do it for both primers forward and reverse, or just one of the primers?????, somebody can help me with that!!!!!
I assume you are using the native ribosomal binding site?
You will have to clone upstream, in-frame with the promoter contained within the expression vector that you intend to clone. This would imply a change in the 5 prime primer but not the 3 prime primer.
I'd need to see your vector and the sequence of your gene of interest in order to be more specific.
-Matt
hi matt, thanks for the reply, i am going to use the p-EYFP-c1 plasmid and i am going to insert the NFATC1 human sequences (pub med ID: NM_172390), but i just going to clone the REL DOMAIN of this gene. i am going to use the NheI restriction site to clone it, but i don't know if i need to put in both primers the restriction sequence of NheI and the ribosomal binding site, i know that i need to insert the sequence in the 5' end but in both primers???, forward and reverse...
i really appreciate if you can help me with that.
thanks
minerva
You will have to clone upstream, in-frame with the promoter contained within the expression vector that you intend to clone. This would imply a change in the 5 prime primer but not the 3 prime primer.
I'd need to see your vector and the sequence of your gene of interest in order to be more specific.
-Matt