RF-Cloning.org Forum

cblank

Member

Registered: 2017-03-13

Posts: 1

Insert size

Hi!

First off, thanks for maintaining such a useful website!

I have a question before I begin RF-cloning. I designed this project because I'm having issues subcloning the old fashioned way, http://www.rf-cloning.org/rf_cloning_project.php?proj_id=2f0a4e5c32429107ef22b15e19d984d1 but before I order the primers I was wondering if there is a limit to the size of insert that works for RF cloning? Would you recommend Gibson or In-Fusion instead (I haven't used either before)? I've had great success with RF-cloning for small inserts and deletions before, but nothing of this size.

Thanks!

Steve Bond

Administrator

Registered: 2014-01-23

Posts: 130

Re: Insert size

Hi there,
The insert size isn't outrageous, and the overall project looks reasonable to me. It won't be as efficient as a smaller insert, but totally doable.
I'd say go ahead with ordering the primers and give it a go. The nice thing is that you can always switch over to InFusion with the same primers if you run into issues.
Best of luck!
-Steve

Hirst

Member

Registered: 2017-06-19

Posts: 1

Re: Insert size

Hi there,
The insert size isn't outrageous, and the overall project looks reasonable to me. It won't be as efficient as a smaller insert, but totally doable.
I'd say go ahead with ordering the primers and give it a go. The nice thing is that you can always switch over to InFusion with the same primers if you run into issues.
Best of luck!
-Steve

What is the maximum insert size allowed?

Last edited by Hirst (2021-10-27 20:25:28)

Steve Bond

Administrator

Registered: 2014-01-23

Posts: 130

Re: Insert size

There isn't a hard rule. Projects with small inserts just tend to be more efficient than projects with larger inserts. Very generally:

<1Kb: Fairly easy
1Kb-3Kb: Getting harder
3Kb-5Kb: Probably going to be difficult
>5Kb: I'd start to consider alternatives.

-Steve