gateway question - pdonr221 (Sep/05/2008 )
Hello,
I am just about to start my gateway cloning and have designed primers to amplify my gene of intrest from the TOPO TA vector with attB sites.
I will be doing the BP reaction to get the gene of interst into the pDonr221 vector. After the BP reaction it is suggested that we use OmniMax 2T1 phage resistant cells.
Currently I do not have these cells. My question can I use normal DH5alpha cells to grow the vectors after the BP reaction. Would I be able to detect the right clones with the antibiotic selection for the vector backbone???
thekid
Dear kid,
You do not need OmniMax 2T1 phage resistant cells for transforming your BP reaction. Transform your BP reaction into DH5 alpha cells ( works perfectly), and screen transformants in Kanamycin. Works fine.
All the best.
Regards
Avinash
http://aviprem.gq.nu
I am just about to start my gateway cloning and have designed primers to amplify my gene of intrest from the TOPO TA vector with attB sites.
I will be doing the BP reaction to get the gene of interst into the pDonr221 vector. After the BP reaction it is suggested that we use OmniMax 2T1 phage resistant cells.
Currently I do not have these cells. My question can I use normal DH5alpha cells to grow the vectors after the BP reaction. Would I be able to detect the right clones with the antibiotic selection for the vector backbone???
thekid
With the gateway system, there are 2 selections, one is the antibiotic and the other with the cells (the ccDB grows only in select cells). So if you dont have the cells, you could still do it with the DH5-alpha cells and use the right antibiotics.