RF-Cloning.org Forum
Anything and everything cloning: Go...
You are not logged in.
- Topics: Active | Unanswered
Pages: 1
#1 Re: rf-cloning troubles » Cloningtroubles when 'swapping' genes » 2014-08-05 06:24:06
I always include a positive control with any transformation 
And it is looking very good, tons of colonies.
Is it possible to electroporate chemically competent cells? Won't I need to make a new batch of cells which are in a different buffer? I think I read somewhere that the salt levels could affect the electroporation process...
#2 Re: rf-cloning troubles » Cloningtroubles when 'swapping' genes » 2014-08-04 07:27:35
I plate the entire 100uL of competent DH5a on ampicillin containing agar plates.
What other sort of cells would you consider? I don't have access many bacterial strains as we always use DH5a in my lab. I also have access to XL-Gold and Top10 cells. If you have any specific strains in mind I can ask around the Microbiology department and see if they have anything I can borrow
#3 rf-cloning troubles » Cloningtroubles when 'swapping' genes » 2014-08-03 19:17:18
- Meyana
- Replies: 6
Hello
I have used your resource to design primers for moving GFP into my destination vector while simultaneously removing a puromycin gene, thus ending out with swapping GFP for puromycin. I am working in a big vector (13kb).
I am able to generate the megaprimer without any troubles (purify on gel). However, after the secondary PCR, DpnI treatment, and heat-inactivation, I do not get any colonies when transforming competent DH5alpha cells. On my first attempt I tried to run the secondary PCR on a gel, but I didn't see anything and I read somewhere that it was probably not going to show up on a gel, so I have skipped this the last attempts.
I have redone everything several times now, but still no luck.
I am using the Phusion polymerase with 30s/kb elongation time. I have tried both the 2- and 3-step secondary PCR with the recommended ng's of vector and megaprimer suggested by the website.
Are there any suggestions to what I can change/try to do next?
Pages: 1