RF-Cloning.org Forum

copperD

Member

Registered: 2016-01-26

Posts: 1

trouble with 9 kbp plasmid

Hi all,

I've been doing RF cloning for quite a while. I always worked with vectors of an final length of 6 kbp without any major issues. With 9 kbp I run into troubles. I end up without colonies. I'm pretty much sticking to the literature in my protocol. In the primer design, I must stick to a certain spot. So, the overhangs of the megaprimer a directly adjacent on the parental vector. I cannot leave out any bp in between. Could this also be an issue?
Any comment appreciated. Thx

Chris

Steve Bond

Administrator

Registered: 2014-01-23

Posts: 130

Re: trouble with 9 kbp plasmid

Hi Chris,
The fact that your 6 Kb constructs are working is great news, because it means you are doing things right from a technical sense. So there is probably something going wonky on the design side of the 9 Kb construct. Did you use the website to design it? It's much easier for me to help troubleshoot if I can look at it directly, instead of trying to work from your description. I can search the backend for past projects if you did design it on the site, I just need something to search for.
-Steve