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!
I wish to ask further on the primer designs, I read the protocol but I'm still kinda confused and uncertain of my own understanding. I couldn't find someone to explain and help me. So, if it is possible, will anyone be very helpful to explain the primer design for in-fusion cloning.
1) Is it possible to create primer for six fragments/ gene-of-interest using the in-fusion primer tool online. From the page, there are capacities for 4 fragments only.
2) If it is not possible, what are the alternatives to design the primer for in-fusion cloning?
3) What are the differences of having 5' overhangs, blunt ends, 3' overhangs in designing the primers?
4) In what situations are more favorable to use universal primer or specific-designed primers?
Thank you in advance for the help!
3) these are different depending on the ends you want your restriction sites to have. Some restriction enzymes leave blunt ends, while others have overhangs, which can be either 3' or 5'.
4) Depends on what you want to know, and the purpose of your cloning.