gateway cloning - (Dec/11/2007 )
Hi, all.
I am a new comer here and thanks for your kindly help.
Last two months I focus on gateway cloning and I did not get anything until now, so i need your precious suggestions.
First i try to reconstruct an Entry vector with pQE9 expression vector. Why did i construct pQE9 expression vector into an Entry vector? because I want to do site-directed mutagenesis of my gene and then test the activity of the encoding protein. If the mutant protein has a high activity, i will transform this mutant sequence into a Bt shuttle- vector and then express the mutant gene in Bt host strain.
I clone the fragment of attL1+ccdB+attL2 from the Entry vector(invitrogen), then i inserted this fragment into pQE9 expression vector by SphI and HindIII digestion sites. Then i digested this recombinant vector by SalI and NotI, and then inserted my gene into the digested recombinant vector. Unfortunately, problems appeared after I sequence analyzed the second recombinant vector: a, I can not find the ATG of my gene and some bases of the att sequence; b, I got a fragment that i can not tell where it is from?
Thank you very much for your assistance and i am looking forward to your precious response.
why there is no person response me?
I am a new comer here and thanks for your kindly help.
Last two months I focus on gateway cloning and I did not get anything until now, so i need your precious suggestions.
First i try to reconstruct an Entry vector with pQE9 expression vector. Why did i construct pQE9 expression vector into an Entry vector? because I want to do site-directed mutagenesis of my gene and then test the activity of the encoding protein. If the mutant protein has a high activity, i will transform this mutant sequence into a Bt shuttle- vector and then express the mutant gene in Bt host strain.
I clone the fragment of attL1+ccdB+attL2 from the Entry vector(invitrogen), then i inserted this fragment into pQE9 expression vector by SphI and HindIII digestion sites. Then i digested this recombinant vector by SalI and NotI, and then inserted my gene into the digested recombinant vector. Unfortunately, problems appeared after I sequence analyzed the second recombinant vector: a, I can not find the ATG of my gene and some bases of the att sequence; b, I got a fragment that i can not tell where it is from?
Thank you very much for your assistance and i am looking forward to your precious response.