RF-Cloning.org Forum

Meyana

Member

Registered: 2014-08-03

Posts: 3

Cloningtroubles when 'swapping' genes

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?

Last edited by Meyana (2014-08-03 19:22:03)

Steve Bond

Administrator

Registered: 2014-01-23

Posts: 130

Re: Cloningtroubles when 'swapping' genes

Have you been plating the entire transformation reaction, or tried transforming ultra-comp cells?
-Steve

Meyana

Member

Registered: 2014-08-03

Posts: 3

Re: Cloningtroubles when 'swapping' genes

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

Jan

Member

Registered: 2014-04-15

Posts: 7

Re: Cloningtroubles when 'swapping' genes

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

I am not an expert on RF cloning, but did you check your competent cells themselves? Transformed them with something you know that should work? Just to see the cells themselves are ok?
Sometimes this is a problem.

Top10 cells , you can try this too.

Another approach: electroporation rather than chemical transformation. Electroporation has a higher efficiency

Steve Bond

Administrator

Registered: 2014-01-23

Posts: 130

Re: Cloningtroubles when 'swapping' genes

I agree with Jan that you should get a positive control working before trying anything else. Just the parental should be fine
That said, it's a big plasmid you are working with, so I'm not surprised that the reaction is running at low efficiency. If you can electroporate, that would be great, or alternatively get hold of some high competency (10^8-10^9) cells. Any standard strain should be fine (as long as it's appropriate for your given construct).
-Steve

Meyana

Member

Registered: 2014-08-03

Posts: 3

Re: Cloningtroubles when 'swapping' genes

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...

Steve Bond

Administrator

Registered: 2014-01-23

Posts: 130

Re: Cloningtroubles when 'swapping' genes

Ya, electro-comp cells and chem-comp cells are two different beasts.