In-fusion HD cloning - (May/30/2013 )
Is there anyone who used In-fusion HD cloning kit to clone a large fragment?
I usually do cloning with TOPO vector (TA cloning) and then transfer the target sequence from TOPO vector into pGL3 vector by sub-cloning.
If we use In-fusion cloning method, do we have to do cloning with TOPO vector and then sub-cloning with pGL3 vector? Or we just insert the target sequence directly into pGL3 vector?
Thank you for your opinion.
I am currently using InFusion to clone a large fragment!
You don't have to do subcloning. The key with InFusion is that there must be 15bp overhangs on each end of your insert. These 15bp are homologous to each end of a linear vector (such that ligation will produce circular DNA). So you must make primers that add these 15bp homology to your fragment.
If you already have the fragment in your pGL3 vector, you can just make the primers and PCR it out of pGL3.
Do you know any program or website for designing primer for in-fusion cloning?
I haven't found any program or website to do it, no. It just took a lot of planning.
If you google 'InFusion HD cloning,' find the user manual pdf... page 8 has a guide on how to design the ends with 15bp homology.
Edit: Here, I attached the pdf!