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designing primers for cloning - designing primers for cloning (Mar/26/2007 )


I am trying to clone a 1.1 kb gene into pEGFP N1 vector (in MCS). The same promoter works for both the MCS and EGFP protein enabling a fusion protein unless a stop codon intervenes. MCS is a few Bases upstream of EGFP. My gene of interest does not have Eco RI and Bam H1 site, which needs to be incorporated. I am also incorporating a Kozak consensus sequence in my forward primer for amplification of gene of interest.

Is there anything else that I need to look at?




Make sure you have enough bases when you begin your primers so that the enzymes can digest properly. I usually add 5 -6 bases before the beginning of the restriction site, followed by the priming region. Also make sure, the sites are unique in the vector. try to create all cloning steps including the final vector virtually and then go ahead with the ordering part. it helps.

good luck !!!