RF-Cloning.org Forum

vsustar

Member

Registered: 2014-07-01

Posts: 2

RFP + gene of interest RF cloning into vector in place of GFP ?

Hello everyone!

I'm new here. I've created a new thread for this topic, to make it cleaner and maybe get more attention (and help )

So the problem is the following:

I have a plasmid pIRES2-GFP - it can express two genes; one construct inserted in MCS and also GFP positioned after IRES. http://www.addgene.org/vector-database/3178/

I'd like to insert one gene (which contains also CFP) into MCS (with unique restriction ligation sites). In place of GFP I would like to insert RFP+1700 long gene (5ptase) with restriction free cloning. See image: http://oi58.tinypic.com/ayvh1k.jpg

The RF would be performed first, so GFP, CFP and RFP wouldn't interfere.

Now the question is; GFP has some identical sequence to RFP on 5' and 3' (about 22 bp). I could use the one on the 5' end in my advantage by primer construction.
The GFP-RFP identical sequence on their 3' end however may cause me some problems, since I would like to insert also 1700 bp of FKBP-5ptase after RFP as well not just RFP. Please see the picture: http://oi57.tinypic.com/14tc8zq.jpg

Does anyone have any suggestions? Should I be worried about this interference and maybe solve it by first RF cloning RFP in place of GFP and then RF clone FKBP-5ptase part?

Any help would be greatly appreciated!

Steve Bond

Administrator

Registered: 2014-01-23

Posts: 130

Re: RFP + gene of interest RF cloning into vector in place of GFP ?

Hi vsustar,
This could be a tricky one, but maybe for a reason you haven't considered.

First, while your insert is fairly long, I think it should be fine. In my mind, the shared sequence between RFP and GFP will actually be beneficial on both the 5' and 3' end, because it's going to stabilize binding of the megaprimer along that whole stretch of sequence. There is enough insert after the RFP to make a loop and stick back down to the plasmid, although you might want to add another couple bases to the non-RFP side of the primer just to be safe.

What might actually give you grief is the IRES. As I'm sure you know, they form some interesting secondary structure, and I've ran into problems when trying to clone in or near them. All I can really recommend is that you make sure your primers' melting temps are in the mid 60s, and keep your annealing step hot. All that said, I have successfully cloned an IRES into pEGFP before, and then plunked a >1kb insert down right beside it. I say go for it!

Feel free to send me a private message with a link to your project before firing it off for synthesis. I can probably give a little more feedback if I see everything together in the main interface.
Take care,
-Steve